#microcompartment

Biology concepts – archaea, bacteria, domains of life, hydrothermal vent ecosystem, chemosynthesis

What is a bigger mistake – to overestimate or to underestimate? If you overestimate someone, you may be disappointed with the result. If you underestimate, you may never realize what they are capable of accomplishing. What is more, your underestimation may cause you to miss incredible things already taking place.

Underestimate the power and importance of

wee small things at your peril. The atom holds

extreme amounts of energy, and we depend on

the tiniest of prokaryotes for our survival on Earth.

It would be a mistake to underestimate the grit and power of some of nature’s smallest organisms. We could talk about this for months, but why don’t we stick to the discussion of prokaryotes and their ability to get along without conventional organelles that we began last week.

We can go farther in praise of the prokaryote by looking at how some of them manage to live in the most inhospitable environments; places that would kill us in seconds, or at least we hope they would. These are the “extremophiles;” the name makes them sound like Saturday morning cartoon superheroes.  For example, Thermococcus gammatolerans is the most radiation tolerant organism on Earth. It can laugh at gamma radiation levels 100x higher than other resistant organisms, even though it lives at the bottom of the sea.

As a result of the molecular biology revolution, many of the extremophiles are now called Archaea (Greek for “ancient”) or archaeabacteria, a completely group of organisms. Archaea are older than bacteria, and but they have some similarities to bacteria. Archaea are generally smaller than bacteria, but the cell wall of most archaea looks just like that of Gram+ bacteria. This is a thicker cell wall than that of Gram- bacteria, and takes up the Gram stain, hence the name Gram+.

The archaea cell wall is thick, and is contiguous with the cell membrane, 

like that of Gram+ bacteria. Gram- bacteria have thinner walls and 

they have a periplasmic space between the wall and the membrane.

Just looking at archaea and bacteria through a microscope makes it hard to tell the difference between these two distant relatives. It is at the molecular level that most of their differences become apparent. The way that archaea make RNAs is more eukaryotic than bacterial and while they both have cell walls, the lipids that make up archaeal membranes are quite different. Archaea lipids are hydrocarbon based, not fatty acid based like those of eukaryotes and bacteria. Also, archaeal cell walls lack the peptidoglycan that is characteristic of bacterial cell walls. Peptidoglycan synthesis is a common target of antibiotics, like penicillins, cephalosporines, and vancomycin.

This last difference might work out O.K. for us as humans. Not a single disease can be attributed to an archaea – yet. This is a big exception. Every other group of organisms on Earth has at least some members that can do humans harm, even if only inadvertently. Fungi, protozoa, bacteria, even plants can all cause us harm. One study says it is unlikely that we have just missed disease-causing archaea. About 0.38% of bacterial species cause disease, so if diversity in archaea is similar to that in bacteria, we should have found about 20 disease causing archaea by now.

Gum disease (periodontitis) has an outside chance of having an archaeal cause, but the evidence is sketchy. In a couple of studies, the presence of archaea in the mouth has correlated with gum disease; if archaea were present, then there was disease. Also, higher archaea number correlated to more severe disease. But archaea were only present in 1/3 of all cases of periodontitis – this is not good evidence to say archaea are the cause of periodontitis. This is the closest we have come to finding an archaeon with an anti-human bent.

Some archaea are thermophiles (heat loving); they don’t just like it hot, some require it really hot. Many thermophiles live in near undersea hydrothermal vents, where heat from the Earth’s mantle and core escapes into the ocean; basically ocean volcanoes.

The hydrothermal vent is an ecosystem that one

would be hard pressed to call home. Varies from 700˚C

to 4˚C, it is acidic, toxic, and radioactive. Yet many unique

prokaryotic and eukaryotic organisms live nowhere else.

To each his own.

Near a thermal vent, the temperature can reach 400-410˚C (700-720˚F) . The water doesn’t boil because of the great pressure exerted on it by all the water above it. No eukaryotic organism can survive at these temperatures, but thermophiles like T. gammatolerans do just fine. The hydrothermal vents pour out high levels of gamma type ionizing radiation from deep in the Earth, so it is handy that this archaeon is a multi-extremophile.

Only a few feet away from the vent the temperature of the ocean bottom will remain near freezing, about 4.5˚C. Other archaea (and some true bacteria) thrive in this cold environment. Called psychrophiles, cold tolerant archaea have cell walls that resist stiffening in water that is even below freezing temperature, and can fill there cytoplasm with anti-freeze proteins (AFPs; they create a difference between a solution melting point and its freezing point, called thermal hysteresis).

Between these two extreme environments, you can find quasi-conventional animals. As the hydrothermal vent water gives up its heat to the surrounding ocean, it creates an area that holds a temperature of about 10-15˚C. Many interesting animals have been found in this area, including the yeti crab and tube worms. Data from January 2012 describes a pure white octopus found at a depth of 2,394 meters. At this depth there is no light, so the octopus has no need for the elaborate camouflage mechanisms of color and texture. This octopod may represent a new species, but other white, vent-dwelling octopuses have been described previously, just not this far south.

This is the yeti crab (Kiwa hirsute). It is white because it lives

in the dark. It is furry because……..well, it makes the name

appropriate. Actually, the setae (hairs) contain bacteria that

may act to detoxify the water from the hydrothermal vents

where it lives. And it isn’t really a crab either, but I’m not

going to tell it so.

Ultimately, even these animals depend on the archaea for survival. No photosynthetic producers can survive at these depths, so the food chain starts with the chemosynthesizing prokaryotes, particularly those that use hydrogen sulfide to produce energy. Hydrogen sulfide is a major constituent in the hydrothermal vent output…. and would kill us quickly by binding to the enzymes in our mitochondria that perform ATP synthesis.

Some animals, like snails, eat the chemosynthesizing prokaryotes directly, while others predate the snails, etc. On the other hand, tube worms (Riftia pachyptila) get their energy directly from thermophilic proteobacterium that live inside the worm in a symbiotic relationship.

Other archaea live in high salt environments, like in the Dead Sea or the Great Salt Lake. They must be lonely, because given the high salinity, they are the only things living there (Water, Water Everywhere, But….). On the grosser end of the scale, some archaea thrive in human sewage plants, working well in environments without oxygen and high nitrogen contents.

Archaea have also been found in natural asphalt lakes, like near the La Brea region of Trinidad and Tobago. With toxic gases, high temperature, and practically no water at all, it was surprising that scientists found so many different kinds of prokaryotes, including several types of archaea. These 2010 findings suggest that life on other planets might not necessarily depend on water – that would be one heck of an exception!

But not all archaea are extremophiles, and they turn out to be much more common than we had thought. This isn’t just a numbers game, it turns out that we have been underestimating their effects on our lives all along. For instance, nitrogen fixation is crucial for crop production. A 2006 study by Schleper et al. in Norway suggests that there are many more ammonia oxidizing archaea in the soil than there are nitrogen fixing bacteria.

Archaea are responsible for much of the primary production that 

occurs in the soil and in the water. Just the methanogenic 

archaea alone are responsible for nearly 2% of all the carbohydrates

produced on Earth. Archaea contribute to the primary production 

of every ecosytem.

Further, current evidence suggests that archaea may represent 25-84% of all primary production (creation of carbohydrates and other organic compounds from inorganic carbon sources, whether by photosynthesis or chemosynthesis) in the upper layers of seawater. Primary production is the beginning of every food chain, so ultimately all of our food depends on archaea as well. To bad that we have been underestimating our dependence on these oldest of life forms. Who knows what our effects our life choices have been having on them all these years.

On the other hand, not all extremophiles are archaea either. Thermus aquaticus is a bacterium that lives in hot sulfur springs and geysers. It is a chemosynthesizing bacterium that has become important in molecular biology. Since its enzymes can tolerate high temperatures, it is useful for replicating DNA sequences in the lab using the polymerase chain reaction. One step in this reaction requires high temperature and would kill most other enzymes. 

Amazingly, this PCR technology and T. aquaticus polymerase has been crucial for helping us see how important the archaea have been in our evolution. In 1977, scientists Carl Woese and George Fox began DNA sequencing of some the extremophiles. They recognized that archaea were very different from eubacteria. The two groups must have diverged long long ago.

The three domains of life are shown here. The length of line shows 

the evolutionary distance between domains. You can see that 

Archaea are more like us than are the bacteria. You can’t tell from 

this chart, but Archaea are older too. They are the roots

of our family tree.

It turns out that Archaea are as closely related to eukaryotes as they are to eubacteria. This stood science on its ear. Up to this point, scientists had been arguing as to whether there were four, or five, or six kingdoms. Now they had to impose a higher classification which superseded all the kingdoms.

Woese’s evidence has led us define to the three domains of life. One domain is the eukaroytes, all the cells with a nucleus (with exceptions, but we can talk about those later), with linear chromosomes instead of one circular piece of DNA (again with exceptions), and with organelles. The second domain is the archaea and the third domain is the bacteria. Six kingdoms follow from these domains; archaea, bacteria, protista, fungi, plantae, and animalia.

Archaea, bacteria, and eukaryotes; we have shown that they are all different, and yet they all developed from some single precursor cell. Next time we will see if our discussion to this point gives us a roadmap to get from that ancient first cell to us.

Rogers, A., Tyler, P., Connelly, D., Copley, J., James, R., Larter, R., Linse, K., Mills, R., Garabato, A., Pancost, R., Pearce, D., Polunin, N., German, C., Shank, T., Boersch-Supan, P., Alker, B., Aquilina, A., Bennett, S., Clarke, A., Dinley, R., Graham, A., Green, D., Hawkes, J., Hepburn, L., Hilario, A., Huvenne, V., Marsh, L., Ramirez-Llodra, E., Reid, W., Roterman, C., Sweeting, C., Thatje, S., & Zwirglmaier, K. (2012). The Discovery of New Deep-Sea Hydrothermal Vent Communities in the Southern Ocean and Implications for Biogeography PLoS Biology, 10 (1) DOI: 10.1371/journal.pbio.1001234

For more information and classroom activities on archaea, hydrothermal vents, chemosynthesis, and domain/kingdoms, see:

Archaea and extremophile bacteria –

http://www.ucmp.berkeley.edu/archaea/archaea.html

http://www.lessonsnips.com/lesson/extremophile

http://euarch.blogspot.com/

www.sinauer.com/pdf/Staley2e_Ch17.pdf

http://www.windows2universe.org/earth/Life/archaea.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Archaea.html

http://www.ucmp.berkeley.edu/archaea/archaeamm.html

http://serc.carleton.edu/microbelife/extreme/extremophiles.html

http://science.nasa.gov/science-news/science-at-nasa/2008/07feb_cloroxlake/

http://www.bath.ac.uk/cer/

http://www.physorg.com/news203835088.html

http://www.eoearth.org/article/Extremophile?topic=74530

http://baliga.systemsbiology.net/drupal/education/?q=content/lesson-3-introduction-extremophiles

http://www.lpi.usra.edu/education/fieldtrips/2007/activities/

http://www.lpi.usra.edu/education/fieldtrips/2007/resources.shtml

http://www.pbs.org/wgbh/nova/nature/lives-of-extremophiles.html

http://www.tes.co.uk/teaching-resource/Extremophile-lesson-6069543/

Hydrothermal vents –

http://www.dlese.org/library/query.do?q=hydrothermal%20vents&s=0&setVocabState=re&setVocabState=gr&setVocabState=ky&setVocabState=cs&setVocabState=su&gr=&re=&cs=&ky=&su=

http://www2.vims.edu/bridge/search/bridge1output_menu.cfm?q=expedition

http://www.pbs.org/wgbh/nova/teachers/activities/2609_abyss.html

http://oceanexplorer.noaa.gov/explorations/06fire/background/edu/lessonplans.html

http://www.thirteen.org/edonline/ntti/resources/lessons/s_dive/index.html

http://www.nationalgeographic.com/xpeditions/lessons/07/g35/seasvents.html

www.msc.ucla.edu/oceanglobe/pdf/Deep.../Deep_6_Vent_Web.pdf

http://www.montereyinstitute.org/noaa/lesson05.html

http://www.botos.com/marine/vents01.html

http://2011.polarhusky.com/logistics/oceans/geology/hydrothermal-vents/

Chemosynthesis –

http://2011.polarhusky.com/logistics/oceans/geology/hydrothermal-vents/

http://www.ceoe.udel.edu/deepsea/level-2/chemistry/chemo.html

http://www.divediscover.whoi.edu/vents/light.html

http://www.montereyinstitute.org/noaa/lesson05.html

http://www.bigelow.org/foodweb/chain4.html

http://oceanexplorer.noaa.gov/edu/learning/player/lesson05/l5ex1.htm

http://www.ehow.com/info_8060311_primary-production-activities.html

http://www.thegateway.org/browse/38643

http://serc.carleton.edu/sp/erese/activities/carbon-cycle-femo.html

Domains/kingdoms -

http://biology.about.com/od/evolution/a/aa041708a.htm

http://faculty.plattsburgh.edu/neil.buckley/concepts%20in%20biology/lecture6domainsandkingdoms.htm

http://www.fossilmall.com/Science/Domains.htm

http://www.ucmp.berkeley.edu/alllife/threedomains.html

http://sciencespot.net/Pages/kdzbioclass.html

http://www.kidsbiology.com/biology_basics/classification/classification1.php

http://www.wisegeek.com/what-is-biological-classification.htm

www.lessonsnips.com/docs/pdf/taxonomy.pdf

Biology Concepts – prokaryotes, simplicity, complexity, organelles, microcompartments

Not everything new is better; new doesn’t

necessarily mean improved. Remember the “New” Coke debacle?

Newer is better, right? Everything old is simple and plain. Back in the good old days, you had to read a book, but today you can browse the internet and pick from 8000 songs while you drive to the superstore to pick up a Kindle. Today is faster. Better. More complex?

How about living in 1800. Could you catch and kill your dinner with a trap of your own making, followed by gutting and dressing it on the back porch of a house you built with your own hands, while you try to keep your entire family from being eaten or dying from an infected scratch?  Now whose world seems complex!

This same belief has been applied to forms of life. Bacteria are old and simple; we are new and complex. Plants and animals can do millions of things that bacteria can’t, because they are so simple and primitive and we are so high tech, biologically speaking.

But it is a mistake to call bacteria simple or primitive. They may not have all the bells and whistles that eukaryotic (eu - true and karyo = nucleus) cells have, but they have survived much longer than other life forms, and they outnumber us by billions. There are more bacteria in a handful of rich soil than people who have ever lived on Earth. So don’t confuse complexity with success.

A cursory look at bacteria would suggest that they are indeed simple. They are bags of chemicals, without the complex organelles that mark eukaryotic cells. Plus, they're small; the whole organism is just one cell. They have just one chromosome and fewer genes than eukaryotic cells. It would be easy to see them as simple.

Even at the biochemical level prokaryotes (pro = primitive) appear simple compared to eukaryotic cells. Our more modern cells aren’t satisfied with just making more proteins, they also modify many of these proteins, adding carbohydrates, acetyl groups, phosphate groups, sulfur groups, etc. These post-translational modifications (after peptides are translated from mRNA messages) are crucial for different functions and for interactions with messengers and DNA.

Histones are protein complexes that help DNA to coil up into tight  configurations. But DNA it is tightly packaged, it is hard for  individual genes to be transcribed and made into protein. Histone  acetyltransferases are enzymes that add acetyl groups to the histones  and open DNA to be read. Histone deacteylases do the  opposite, they add acetyl groups and cause the DNA  to tightly coil.

The exception here is that less than a decade ago scientists found that many prokaryotes also do some kinds of post-translational modifications, includingphosphorylation and acetylation. Acetylation, the addition of a -COCH3 group to a molecule, is important in eukaryotic cells for several reasons, not the least of which is in determining which DNA is open to be replicated or transcribed (copied to mRNA).

Data from 2004 was the first to show that prokaryotes can carry out phosphorylation (addition of PO3 groups) to proteins. What is more, acetylation and phosphorylation are reversible modifications, so an additional layer of complexity is added. Prokaryotic proteins have one function when modified and another when not modified, just like modification of eukaryotic proteins. Sounds like prokaryotes have more going on than we thought.

Prokaryotes are the real success stories of life on Earth. They can do things some things eukaryotes can’t do (more on this next time). Even more amazing, every deficit we have said they have - they can’t do this, they don’t have those – can be seen as a reason they are more amazing.

Prokaryotes are single celled organisms, so they have less specialization. But this means that the cell has to carry out every function that the organism needs. Could your fat cells produce antibodies and kill off protozoan invaders? I think not. We also poke fun at prokaryotes because they don’t have organelles; but this means they have to find a way to do all their chemistry in one big open environment, much more difficult .……….or maybe not.

That classic rule of biology, "eukaryotic cells have organelles and prokaryotic cells don’t," may not be completely true. This would be a big exception.  Evidence shows that many kinds of prokaryotes do have local environments, called microcompartments. We have all been living a lie!

The most studied of the microcompartments is the carboxysome. This hollow shell, first described as far back as 1956, holds enzymes (RuBisCo, see When Amazing Isn’t Enough) that many prokaryotes use for carbon fixation. Photosynthesis is the most obvious type of carbon fixation, where carbon in a gas form (CO2) is converted to carbon in an organic, solid form (carbohydrates).

Carboxysomes as seen by electron microscopy. They really

do look geometric. The faces and corners are specific groups

of proteins, and hold the enzymes inside the microcompartment.

There are minute pores where the proteins come together

to let reagents and products move in and out of the carboxysome.

RuBisCo is a fairly inefficient enzyme, so sequestering it with its substrate inside a microcompartment works to increase the production of energy. Doesn’t this sound a lot like one of the key reasons for the development of organelles – the bringing together of reagents for increased efficiency of reactions?

But it is not just photosynthetic bacteria (cyanobacteria) that use carboxysomes. Many other autotrophic bacteria (auto = own and troph = food) use carboxysomes to fix carbon during chemosynthesis. Chemoautotrophs, for instance, are organisms that use chemical energy rather than sunlight energy to fix carbon.

In many prokaryotes, the oxidation of hydrogen sulfide or ammonia (a nitrogen containing compound) provides the energy for producing organic carbon; Thiomargarita namibinesis from our posts on giant bacteria uses sulfur for chemosynthesis. But there are also organisms that use the energy from the production of methane to drive carbon fixation. You have undoubtedly had experience with intestinal prokaryotes that produce methane gas (methanogens) – don’t try to say you haven’t.

The carboxysome (as a model of many microcompartments) is not a membrane bound bag as organelles are in eukaryotes. Carboxysomes are more like soccer balls made of protein, but in this case they hold a rigid polyhedral form and don’t get bicycle kicked into a prokaryotic net by Pele.

Each face of the shell is made up of a two dimensional polymer of protein hexagons. However, as architects will tell you, this is a difficult shape to close using only hexagons, even with 10,000 of them, like the typical carboxysome has. Soccer balls and the dome at the Epcot Center use strategically placed pentagonal faces that allow for the turning of the hexagonal faces and a closing of the compartment (see cartoon above).

These are cartoons showing the structure of a

carbon fullerene (right) and a carbon nanotube

(left). Each green sphere represents a carbon atom.

These structures are very strong, like for making

bicycle helmets. They may also become useful for

things like space elevators, nanoelectrical circuits,

and solid lubricants.

We have used this hexagonal and pentagonal combination for decades, but it was identified in bacteria less than five years ago. This arrangement is also seen in viral protein coats, as well as in carbon fullerenes, which are superstable carbon nanostructures described in 1985 and named for the inventor of the geodesic dome, Buckminster Fuller.

Could this be the exception – nature stealing an idea from humans? Probably not, I’m guessing Dr. Fuller independently happened upon the same solution that nature had worked out millions of years ago – but it took a heck of an intellect to recognize a good thing.

It might be lucky for us that Fuller’s domes had us looking for this combination in other areas. Carboxysomes are present in up to 25% prokaryotic pathogens (disease causing organisms), and current research is aiming to disrupt the formation of the hexagonal/pentagonal compartments as a way to kill, or at least slow down, the microbes. So many prokaryotic pathogens are developing resistance to traditional antibiotics that a new approach will be heartily welcomed.

There are other microcompartments besides the carboxysome. The bacterium Clostridium kluyveri is proposed to have a metabolosome compartment for the conversion of ethanol into carbohydrates. Furthermore, Salmonella enterica, is capable of producing two different metabolosomes; one for propane-1,2-diol and one for ethanolamine, for conversion of these substrates into energy-containing carbon sources. The evidence of these additional microcompartments makes one wonder just how many different species of protein shelled microcompartments there may be. To investigate this question, a group from UCLA recently published a study using comparative genomics (comparing genes of similar and dissimilar organisms to find groups of genes of similar function) to point out possible enzyme pathways that may be sequestered in microcompartments. Their late 2012 study suggests that new types of microcompartments for different types of propanediol metabolism, and the identification of microcompartments in organisms for which they were previously unknown, like mycobacteria. The genomic evidence also suggests new types of protein shells, differing compartments being used for differing variants of enzyme function.

It is in these final examples that we see a more concrete purpose for the microcompartment. During the metabolism of alcohol, propane-1,2-diol, or ethanolamine, a compound called acetaldehyde is formed. This is a toxic product that needs to be converted to acetic acid in rapid order to avoid toxicity to the cell. By isolating the acetaldehyde in the metabolosome, S. enterica improves its own living conditions. This is also important to us humans.

This is not a before and after picture for an embarrassing

karaoke incident. This is a demonstration of the facial

flushing reaction when a person has an ALDH2 mutation, and  can’t metabolize alcohol efficiently.

Many Asians and Ashkenazi Jews have a mutation of the acetaldehyde dehydrogenase (ALDH2) gene that produces the enzyme that rids the body of acetaldehyde after the consumption of alcohol. The mutation produces a poorly functioning enzyme, so acetaldehyde builds up in their systems and causes a facial flushing reaction. If both ALDH2 genes (one from mom, one from dad) are mutated, the person gets violently ill from consuming ethanol. As you might imagine, populations in which this mutation is prevalent have very low rates of alcoholism.

So we have the exception that prokaryotes are not really without organelles; theirs just look different. Could you guess that the exception goes the other way too? Well, it does. The nucleus of eukaryotic cells works with microcompartments that allow certain things in and out, but keep your DNA inside the nucleus.

The pores of the nucleus (Cells Are Great Multitaskers) are complex openings made up of many proteins. Why? Nuclei could just use receptors to allows certain things in or out, similar to the system used by the cell plasma membrane. But evolution went with a more complex solution.

The vault complex is made of 78 identical protein chains.

One chain is shown in white. Together, they form a

microcompartment that is crucial for our nucleus function.

There is a protein microcompartment called a vault complex that works with the pore complex. This is a highly regulated way of moving RNAs and ribosomes (made in the nucleolus which is inside the nucleus) out of the nucleus, while keeping your DNA inside. I don’t think it is a hard concept to grasp that you cells are happier when your DNA stays inside the nucleus; do you keep your valuables on your front lawn?

Next time we will see how the nucleus, its pore complex, and its microcompartment carriers helped us make the jump from prokaryote to eukaryote. The nucleus is a later evolutionary development, but it still uses a prokaryotic system. This is clue that helps us investigate our cellular family tree. 

Jorda, J., Lopez, D., Wheatley, N., & Yeates, T. (2012). Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria Protein Science DOI: 10.1002/pro.2196 For more information or classroom activities on bacterial microcomponents, post-translational modification of proteins, alcohol metabolism, or the vault complex, see:

Bacterial microcomponents –

http://www.sciencedaily.com/releases/2008/02/080221152009.htm

http://schaechter.asmblog.org/schaechter/2011/04/beyond-the-bacterial-microcompartment.html?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+schaechter+%28Small+Things+Considered%29

http://www.microbemagazine.org/index.php/06-2010-home/1863-the-carboxysome-and-other-bacterial-microcompartments

www.asm.org/ccLibraryFiles/Filename/.../znw00107000025.pdf

http://www1.cnsi.ucla.edu/arr/paper?paper_id=196039

http://scienceblogs.com/oscillator/2010/03/bacterial_organelles.php

http://www.microbialcellfactories.com/content/10/1/92

Protein post-translational modification –

http://www.piercenet.com/browse.cfm?fldID=7CE3FCF5-0DA0-4378-A513-2E35E5E3B49B

http://themedicalbiochemistrypage.org/protein-modifications.php

http://www.premierbiosoft.com/glycan/glossary/post-translational-modifications.html

http://www.cook.rutgers.edu/~dbm/posttrans.html

http://www.juliantrubin.com/encyclopedia/bioinformatics/proteomics.html

Alcohol metabolism –

http://www.elmhurst.edu/~chm/vchembook/642alcoholmet.html

http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&ved=0CGMQFjAI&url=http%3A%2F%2Fjournals.cambridge.org%2Fproduction%2Faction%2FcjoGetFulltext%3Ffulltextid%3D980664&ei=v4lvT-KgBci-gAfuwonpBw&usg=AFQjCNEf-W1CVy-a75h8Z1s1HVLbtVhkSA

http://science.howstuffworks.com/environmental/life/human-biology/hangover4.htm

http://hamsnetwork.org/metabolism/

http://en.wikipedia.org/wiki/Alcohol_flush_reaction

http://ethemes.missouri.edu/themes/315

Nuclear vault complex –

http://www.vaults.arc.ucla.edu/pages/sci-pores

http://micro.magnet.fsu.edu/cells/nucleus/nuclearpores.html

http://www.ks.uiuc.edu/Research/npc/