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Archive for September, 2011

Free College, Beware the wrath of Zeus

“If my typical man wishes to live fully and completely he must, in his
mind, arrange a day within a day.  And this inner day, a Chinese box in
a larger Chinese box, must begin at 6 p.m. and end at 10 a.m. It is a
day of sixteen hours; and during all these sixteen hours he has nothing
whatever to do but cultivate his body and his soul and his fellow men.
During those sixteen hours he is free; he is not a wage-earner; he is
not preoccupied with monetary cares; he is just as good as a man with a
private income.  This must be his attitude.”  A. Bennett

Ever wanted to become a nuclear physicist? Or take that one course offered junior year, but couldn’t fit it in with the butt load of courses already in your major? Need to learn some new rhetorical skills to debate with some honky online or in a bar? Or, maybe you just have a growing interest in something that sparked your fancy in the news?

Well good, because MIT offers their courses, class notes, homework and tests online for free and open to the public. Of course, they don’t offer credit, but this is a find, nonetheless.  Similar to the Feynmann lectures on physics, class lectures for courses are offered in videos, making access to the material all that much easier.  Study packets offer PDFs of the lecture notes and slides and problem sets to test your mettle.  A person would still have to buy the texts to study from, but it is often possible to collect the same information from online sources as well, such as google books or project Gutenberg.  Having done this myself, it can be troublesome to find exactly what you need, though.  However, for a serious student in search of enlightenment, there is always amazon’s speedy UPS delivery of a cheaply priced used text.

Not only MIT, but many universities have begun offering similar online programs as well as.  This is a trend that is only beginning, as most universities have also incorporated some form of online program in conjunction with ‘brick and mortar’ coursework.  Online discussion groups are an often used system to incorporate new ideas and group-think solutions to problem sets.

The internet offers plenty of other opportunities for self study as well.  Take for example, the Khan academy, which offers tutorial videos on math and science topics from grade school on through college.  K-12 series has problem sets that can be worked out while listening to a lesson.  The problems can be broken down piecemeal in a stepwise format if the material becomes tricky.  Additionally, success and hard work are rewarded beyond personal achievement by winning merit badges, with a system similar to that used in scouting.

The Khan academy is the brain child of Salman Khan, whose wish is to: “provid(e) a high quality education to anyone, anywhere.”  One of the cool innovations is not just using the ‘you tube’  style format, but students and educators alike can use the website to monitor performance and find areas of weak understanding  by charted classroom data.  This goes a step beyond the old fashioned grade cards of yesteryear and implements  better what the controversial No Child Left Behind Act had originally intended: to provide real feedback on student progress.  Clearly, this has implications not only for home schooling, but also for education as a whole, as this method allows personalized access to students performance. Technology has changed the way we communicate and so too must it change the way we teach.  Open, free access to information and materials has become a pedestal of the ‘new learning’ and has enabled those away from educational institutions to continue their education.  

For work related experience, the internet can also be an instructional tool.  This used to be nearly entirely the domain of those offering certificates in IT management or Microsoft products, yet this is not the case anymore.  Consider Protocols online, a website devoted to documenting protocols and procedures of lab techniques, which has a large reservoir of articles detailing general lab procedures in life sciences.  Many of the techniques detailed are now commonplace in today’s modern labs, such as western blotting, electrophoresis and H&E staining, just to name a few.  As with any wiki, it wouldn’t be advisable to use it as your only source of information, but it could be used as a good source of quick information to direct further searches.  Having all the information in an easy to search, easily accessible area reduces the time needed to do research and planning and can streamline preparation time.

Today, every one of us can steal fire from the gods.  Scream it loud from the tops of the highest buildings in the city: “I am Promethius!”

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“Wolf’s liver taken in thin wine, the lard of a sow that has been fed upon grass, or the flesh of a she-ass taken in broth.” –Pliny the Elder

Ring of Inhibition

We’ve come a long way in some ways, at least since “flesh of a she-ass” was considered a specific remedy from disease.  Though, did you know that in early Greece moldy bread was a remedy against infection of wounds?  The mold was presumably a kind of penicillium that produced chemicals inhibiting bacterial growth.  On that fine day in 1928, Alexander Fleming had no idea he would discover something that would change medicine forever.  After coming back from vacation and sifting through the piles of clutter in his office he noticed a culture of staphylococcus in which a blue mold was growing.  The mold had pushed back the staph in a characteristic ring of inhibition.  As the story goes, he hypothesized that the mold must be producing something that retarded bacterial growth.  Penicillium chrysogenum produces the chemical backbone used to produce penicillin, an antibiotic which caused a great leap forward in western medicine.  The effect has been so great that the old killers of yesteryear, the terrible diseases that for ages struck fear in the hearts of man are no longer thought of in the same grave tone and are considered more of a nuisance to public health, a mere trifle in consideration of the far greater concerns of the general populace.  Perhaps the greatest triumph is the reaction of surprise that “people still get that?!”

The back bone of penicillin antibiotics, 6-aminopenicillanic acid, was originally isolated from cultures of Fleming’s Penicillium

Various functional groups can be added to the amino side chain to produce different drugs.

notatum.  It is a secondary metabolic product, released only when the mold is under environmental stress.  Early manufacturing methods were hampered, because finding just the right conditions for the mold to release the chemical proved difficult, thus production was initially low.  When the first human trial in the U.S. began in 1942, doctors used half of the nation’s total production of penicillin to cure him. The medical value placed upon it began a race to massively increase its production. Researchers from both Britan and the US worked tirelessly to not only elucidate the shape of the molecule, but develop better processes for producing it.  In 1943 a nationwide search was held to find better strains from which the B-lactam skeleton could be obtained.  The lucky winner was provided by a moldy cantaloupe from Illinois, which produced many times the amount of penicillin than Flemming’s original.  This strain exceeded expectations and was quickly put to work manufacturing the raw penicillin components.  At the time, it was so prized that during World War II doctors would collect their patient’s urine to isolate and reuse the excreted portion of penicillin.

Today genetically engineered strains manufacture it in large tanks where conditions such as the pH, temperature and diet are all automatically controlled to elicit maximum output (S).  The growth medium is key to the entire process, where the goal is to put them under nutritional strain.  Growth mediums are usually proprietary information, but they typically contain a source of carbon and nitrogen, which are chosen to make them work harder to gain the nutrition they need to survive.  Penicillium notatum prefers the simple 6 carbon sugar glucose, but can also digest a similar sugar called lactose.  This change in sugars forces them to work harder for the same amount energy.  Looking for an edge to outpace their competitors, they produce chemicals like 6-aminopenicillanic acid that impede bacterial growth.  The incubating contents are constantly aerated to allow for maximum growth throughout the tank.  When the growth phase has completed the contents are filtered and the raw product is extracted, concentrated and purified.  The liquid can then be altered chemically and turned into a number of antibiotics by adding functional groups to the base skeleton.

Upon discovery of the antibiotic nature of this compound, researchers put the skeleton through many thousands of reactions in order to find compounds with similar properties.  There are many kinds of penicillin, which differ by the addition of unique functional group.  For example, adding phenyl acetyl chloride to the purified preparation produces benzyl penicillin, or penicillin G, as it is more commonly known.  These new additions can change the properties of the original compound, endowing it with activity against a wider range of bacteria or a specific subset of them.  Penicillin G, for example, is strongest against Gram positive organisms, like staphylococci and streptococci, and gram negative cocci, like N. gonorrhoeae, but has low activity against bacteria that produce enzymes that inactivate the compound, called B-lactamases.  With N. gonorrhoeae, however, there has been increasing resistance of this kind reported and penicillin G is no longer considered the drug of choice to treat them.  According to William Strohl, “Over twenty percent of current isolates of N. gonorrhoeae are resistant to penicillin, tetracycline, cefoxitin, and/or spectinomycin.” (S)

B Lactams usually block the enzyme that ties these pieces together.

With such a wide range of activities against so many organisms one might wonder how they work.  Like all poisons, penicillins inhibit critical life processes in the species attacked. In this case, they inhibit bacterial growth by stopping the repair and replacement of their cell walls.  In affected strains, the cell walls are composed of long chains of six carbon sugars with amino acid side chains that weave the chains together like a net.  The long chains are made by polymerizing N-acetylglucosamine and N-acetlymuramic acid sugars into alternating units.  The side chains are made of repeating amino acid units, which differ per species, but for susceptible species they end with the combination D-alanyl-D-alanine amino acids.  B-Lactam antibiotics are natural analogues of these amino acids, which is to say they can fit into some of the same places as the natural bacterial amino acid piece can.  The antibiotics attach themselves to a special protein (the aptly named ‘penicillin binding protein’) that transports and tethers the D-alanyl-D-alanine amino acids to other chains in the cell wall, blocking its activity(S).  With these proteins blocked the cell wall can’t be replaced or repaired. Lysis results when the cell wall is unable to withstand the osmotic pressure upon it.

It is important to point out that these antibiotics are most active when bacteria are growing and synthesizing cell walls, which is fine for fast growing colonies, like the streptococci.  However mycobacteria, which are famous for causing tuberculosis and leprosy, have a dormant states where they don’t actively synthesize new cell wall components as well as impregnate them with certain lipids that impede the entry of penicillins.  In this context, an attack of this kind would be useless or at the very most a stop gap measure.  Since the colony isn’t completely killed off during treatment, this situation is favorable for the development of antibiotic resistances and the selection of hardy strains of the bacteria.  In a clinical setting these factors complicate issues, with treatments for tuberculosis taking upwards of a year or more to complete.

In a colony of 10^6 bacteria, the probability is that at least one will have resistance to a given antibiotic.  A typical mycobacterial colony will have something around 10^8 members, making the selection of strong, antibiotic resistant strains almost a certainty.  This problem is further compounded by a form of bacterial communication called horizontal gene transfer or plasmid exchange.  This is where a circular strand of DNA, typically encoding a gene producing a resistance, is absorbed by other bacteria allowing the technical means of resistance to spread. This is how bacteria such as the dreaded MRSA have developed and why infections from them are most common in hospitals. In the case of tuberculosis, multi-drug therapies utilizing non-B-lactam antibiotic combinations, like isonaizid-rifampin, can cure 95-98% of cases (S).  The probability that one will have a resistance against both antibiotics is very low and using two will usually kill the bacteria that have a selected immunity to one antibiotic alone.  The other 2-3% typically require specialized treatment and top of the line antibiotics, like moxifloxacin or rifabutin.  However, moxifloxacin resistant strains have been recently identified as a cause for concern (S).

Bacterial resistances usually present via four different routes: Reduced membrane permeability to the drug, production of an enzyme that deactivates the antibiotic (a B-lactamase, for example), a mutation in the PBP site where the antibiotic binds and finally, the creation of a means for drug removal (S).  With penicillins, B-Lactamase production is the most common form of resistance, some of which are highly specific to only one kind of drug (S).  B-lactamases attack the core structure of the penicillin molecule, breaking a critical carbon-nitrogen bond.  Mutations in penicillin binding proteins alter the shape of the binding site, making it harder for the B-lactam structure to fit.  In theory mutations could be great enough to block the penicillin from binding completely.  In practice, however, mutations that cause a PBP to be blocked entirely are often less useful functionally, because these will also block the D-alanyl-D-alanine amino acids they were originally intended for.  Small mutations are more useful to inhibit rather than block the penicillin and keep them from reaching a concentration necessary to inhibit bacterial growth. (S)  Gram negative bacteria usually develop resistances to antibiotic entry by changing the shape of receptors that transport the antibiotic across its membrane.  If the drug can’t pass through the membrane, it can’t attack the cell wall manufacturing machinery.  Another means that changes the membrane permeability is the creation of what is called an efflux pump.  These work to transport the antibiotics back across the membrane after entry.  These are of varied efficiencies, but always work to increase the amount of antibiotic required for efficacy.  Both mutation and changes in permeability are often paired with a B-Lactamase enzyme, making an effective, multi-front strategy for resistance.

Even though we have produced effective weapons against pathogenic bacteria, they have begun to fight back.  They have been largely successful in this regard, calling into question how long the usefulness of these drugs can be maintained.  This has spurred the demand for newer drugs to combat the potential ‘super-bugs’ on the horizon and limited use policies to maintain drug effectiveness in the future.  All in all, penicillin is generally considered non-toxic.  The worst adverse reactions are due to hypersensitivities.  Allergic reactions occur, however, this is a rarity occurring in less than 1% of people and is usually not life threatening (a rash, fever, etc).  Even less have a serious reaction, like anaphylactic shock (0.05% of patients).  In a worst-case scenario, though, desensitization to the drug can be used to effectively treat a given disease.

One thing is for sure is that we will continue to look in awe at the complexity life has to offer.  We hope that new attempts to cultivate and extract new antibiotics will be successful in the future.

{sources available upon request}

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To live and function the human body needs energy, the majority of which is produced by the electron transport chain.  The end products from these enzyme catalyzed reactions are oxidized by the oxygen that you breathe and converted into water.  At the center of this complicated process is an incredible  protein that selectively transports oxygen to tissues in need and releases it on demand.  Hemoglobin is a protein carried by red blood cells that has selective affinity for iron, a metal critical to proper function of the body.  The importance of this protein is exemplified by what happens when it is broken.  Sickle Cell Anemia, for example, is caused by a mutation in the gene that codes for the protein.   In this case, the change of a single amino acid (glutamic acid to valine) damages its functionality such that the average life expectancies of people with this disease are only 42 years in males and 48 in women (S).  During this time, those afflicted also experience a host of quality of life maligning health problems, like chronic pain, increased risk of infection and heart disease and vaso-occlusive crises that occur when the misshapen blood cells block blood vessels.

Considering the structure, hemoglobin is a large protein made of four polypeptide chains, sewn together into a tetramer.  There are 2 β chains and two α chains, each of which having a net-like porphyrin ring system.  In human red blood cells, an iron atom is placed in the center of the ring structure, creating a coordinated system called a heme group.  The iron atom is the star of the system and makes the binding of oxygen possible.  It is in the +2 state and has octahedral symmetry, allowing 6 ligands to bind.  The 4 nitrogen atoms of the ring bond equatorially holding the Fe+2 in place, while from the bottom, a histadine amino acid from the protein super structure locks the Fe+2 atom in place.  The open top position is reserved for O2 binding, but in its absence a molecule of water usually takes its place in normal circumstances.

3D images showing the positions of the porphyrin ring, iron and histidine in the heme group. Nitrogen atoms are in blue, iron in red and carbon in the usual grey-black. (3D images courtesy of http://www.3dchem.com By all means check out the structures for yourself using their tremendously useful java based web tool!)

This same system is common throughout other enzymes and respiratory systems.  In humans, Cytochrome C  of the electron transport chain has a similar structure, except that the 6th (top) ligand is a methionine group.  The protein in this case is designed to transport electrons rather than oxygen molecules.  Myoglobin, which is found in muscles and also used for O2 transport, is structurally different, but utilizes a single porphyrin ring system, rather than four.  Enzymes, such as catalase and peroxidase,  also contain Fe manipulating systems similar to the above.  In other kinds of organisms too, the porphyrin ring is utilized in a variety of situations and complexed to many different metals depending on the conditions and needs of the creature involved.  Photosynthetic plants, for example, utilize the same ring, but the metal is magnesium.  Further, some bacteria are also known to use copper as the porphyrin metal of choice.

To make full advantage of this heme system, the binding of O2 in red blood cells is facilitated by a buffer system.  The concentration of any of the components of it increase or decrease the binding affinity of O2 at the hemoglobin binding site.  Proton (H+), CO2, Cl-, and 2,3-Bisphosphoglycerate (BPG) concentrations all have a role in the binding and release of O2 from hemoglobin.  For example, in tissues where the pH (H+ concentration) is acidic and the partial pressure of CO2 is high, the binding affinity of O2 at the binding site will be lowered and will induce hemoglobin to release its contents.  In the lungs, however, the O2 concentration is high compared to that of CO2, facilitating O2 binding.  Normally, BPG is found in equal amounts to hemoglobin, but in situations where O2 is in short supply this balance is undone by the increased production of BPG.  With BPG, binding to one of the active sites reduces the O2 binding affinity by about 25 times, thus when BPG concentration is high it pushes for a release of O2 into tissues that need it.

The larger Fe staggers the planar porphyrin ring, where as the smaller version fits better, contributing to stability.

Aside from outside effects promoting binding, there are effects coming essentially from the design of the system itself that have to be considered as well.  The question of why iron and not some other transition metal is of special relevance here.  Using iron as the central metal, in this case, yields benefits in terms of performance and binding specificity to oxygen.  Cobalt, for instance, is used in similar systems like those of vitamin B12’s corrin ring, but while both cobalt and iron contain d orbitals that could bind, only iron allows for the perfect balance between size and binding specificity in this system.  While unbound, the Fe+2 atom is just a little too large to fit into the normally planar porphyrin ring.  In this case, it is in a high spin state where the molecular orbitals are further away from the atom, giving it a larger size.  The high spin state is larger, because the Pauli exclusion principle prohibits the atom’s 3d electrons from getting too close to one another due to repulsions.  Thus, additional space is needed to house the electrons in orbitals further away.  However, when the Fe+2 binds to an oxygen molecule the electrons can be shuffled into orbitals that are closer to the atom.  Its orbital symmetry changes to a low spin symmetry and CLICK ! the Fe+2 shrinks just enough to fit snugly into the porphyrin ring system.

A silly, but useful comparison.

This part also contributes to a critically important finding: that the binding of O2 is cooperative, in that the binding of one O2 molecule will facilitate the binding of another until all four spaces are filled.  It has been experimentally determined that the binding energies (the Ka) are increasingly smaller for each molecule of O2 bound to hemoglobin.  This happens, in part, because of the protein’s structure.  When the O2 binds and the Fe+2 clicks into place, it pulls on the histadine residue below it, stretching the other protein superstructure bonds.  This pulls slightly on the other 3 Fe+2-histadine bonds.  Much like those dancing string toys, pulling on the string at the bottom causes the toy’s arms and legs to move in a concerted action, which is similar in a way to the physical reaction of the other binding sites.  The additional pulling on the other histadine residues facilitates binding of the other three, such that the binding energy is progressively reduced with each binding until all four slots are filled.

Other structural contributions also play a large role in binding specificity.  Above the plane of the porphyrin molecule lies another histadine residue that physically blocks the strongest and most effective bonding interactions from occurring.  Since the Fe+2 atom is locked into place, the best bonding interactions would come from ones that provide the most overlap of their molecular orbitals, which are those that are end-to-end.  However, with the histadine in the way, these are prevented from occurring.  This is a good thing, because strong covalent interactions in enzymatic reactions, like those seen in the binding of carbon monoxide (CO) for instance, are usually toxic and are difficult to break under normal conditions.  For head-to-head binding to CO, the interaction is estimated to approximately 1000 times as strong as those between O2, illustrating its toxic potential.  In this case, the CO-Fe binding is still strong, but not so much as to completely block removal.  People who have suffered CO inhalation are often given pure oxygen in an attempt to out pace CO binding and ensure that the person continues to have a supply of oxygen.  This hindered binding is also helpful in normal activity as well, since the binding symmetry to O2 is bent as well it further facilitates it’s release into tissues in need.

Yes, it’s about iron again, but it’s quite interesting stuff.

{sources available upon request}

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