Bioluminescence

In many biological studies it is essential to be able to translate into an observable and measurable signal the specific process under analysis. This is normally possible through the use of assay systems based on radioactivity, photon absorption or photon emission. Radioactivity and photon emission are less and less used nowadays, the first due to the restrictions and cares associated with the use of such hazardous materials, the second due to its low sensitivity.

Photon emission can occur by two different processes- either fluorescence or chemiluminescence. Both processes involve the emission of photons associated with transitions between energy states, but the way the excited state is generated varies. In fluorescence, the absorption of light (i.e. of photons) produces the excited state, while in chemiluminescence this is due to exothermic chemical reactions (see diagram- fluorescence is on the right, luminescence on the left).

Both of these processes have their advantages and disadvantages. Fluorescence (of which the most famous example is probably GFP) normally shows a much stronger signal, as the source of the excited signal are photons which can be introduced in the sample at very high rate. However, this jamming of photons tends to create a high background signal which influences the sensitivity of the assay. Chemiluminescence does create lower intensity signals, but as no photons need to be provided there is hardly any background signal. Whichever of the two systems is used will depend on the machinery required in each assay. If the efficiency of light collection is limited, then fluorescence is the best option, this being the reason why until recently fluorescence was the main assay system used. The development of more sensitive machinery, however, has made the background noise the most important problem to solve, therefore justifying the increased interest in chemiluminescence systems.

Bioluminescence is a form of chemiluminescence that naturally occurs in certain living organisms. The enzymes that catalyse this reaction are called luciferases and their substrates luciferins. Note, however, that these are very general terms as bioluminescence seems to have evolved several times independently. This is why the several existing luciferases have such different molecular structures. In fact, luciferases have been cloned from several different types of organisms, namely jellyfish (Aequorea), sea copepod (Gaussia princeps), corals (Tenilla), click beetle (Pyrophorus plagiophthalamus) and several bacterial species. The most successfully luciferase, however, is the widely used firefly luciferase, from the firefly Photinus pyralis. It is commonly used in its humanized variant (codons optimized for mammalian expression), requiring ATP and magnesium in the oxidation of its substrate luciferin, and yielding a yellow-green light with a maximum luminescence of 560 nm.

Firefly luciferase (FLuc) is normally used as a reporter gene, which may mean very different systems of reporting. The most obvious is, of course, the insertion of the luciferase gene in the same plasmid and under the same regulatory promoter as the gene of interest. In my current department (Gene Therapy department, NHLI), for example, firefly luciferase is the main reporter gene used in CFTR transfections. However, FLuc can report more than the expression levels of transfected genes. As it depends on ATP for its activity, it is ideal in assays of ATP concentration. It can also, for example, be an indicator of how well G-protein coupled receptors (GPCR) work. This is possible by using a cAMP response element (CRE) upstream of the luciferase gene. GPCR activation causes an increase in intracellular cAMP concentration. This increase leads to Protein Kinase A activation, which in turn phosphorylates CRE binding protein. The CRE binding protein will bind to the CRE upstream of the luciferase gene, leading to increased transcription. Overall, an increase in CPCR activity leads to increased luminescence signal. This type of report system, for example, is useful in the study of GPCR agonists. The complexity of the system, however, makes it prone to interference, which can lead to false positive results. To prevent this, a dual-reporter system can be used involving a second luciferase, Renilla Luciferase (RLuc), which is an internal control to detect aberrant data. A dual luciferase system can also be used to detect two different processes or expressed genes simultaneously (as FLuc and RLuc have different spectral maximuns, namely 560 and 480 nm).

As a reporter of transfected gene expression, FLuc shows a few limitations, namely the fact that it is an intracellular protein. This means that measuring of transfected gene expression requires, in in vitro cell culture the lysis of the transfected cells, and in in vivo studies the killing of the animal in order to access the transfected tissue. In patients under clinical trial, luciferase assays imply invasive obtainment of tissues (I am still trying to find out how this is done exactly). This is why the development of new luciferase, namely a secreted luciferase is important. Until now, around 4 types of secreted luciferases have been found, but the only one commercially available for cell supernatant assays is that produced by the copepod Gaussia princeps (see figure). Gaussia luciferase (GLuc) is naturally secreted from cells and used by this animal as a defence mechanism. As Gaussia princeps lives at a depth of between 350 and 1,000 m, the sudden production of light is a good distractive mechanism against dark-adapted predators. As a secreted luciferase, Gaussia is reported to give very good signals when medium supernatants are assayed. The important question right now (at least for me) seems to be if it can be assayed in something more… well, you will have to wait for my year-away talk next year for more information!

References

Fan F., Wood K. (2007). Bioluminescence Assays for High-Throughput screening, Assay and Drug Development Technologies, 5. pp 127-136

Markova S., Golz S., Frank L., Kalthof B., Vysotski E. (2004). Cloning and Expression of cDNA for a luciferase from the marine copepod Metridia longa, The Journal of Biological Chemistry, 5. pp 3212-3217

Roda A., Pasini P., Mirasoli M., Michelini E., Guardigli M. (2004). Biotechnological applications of bioluminescence and chemiluminescence, Trends in Biotechnology, 22. pp 295-303

Serganova I., Moroz E., Moroz M., Pillarsetty N., Blasberg R. (2006). Non-invasive molecular imaging and reporter genes, Central European Jornal of Biology, 1. pp 88-123

Tannous B., Kim D., Fernandez J., Weissleder R., Breakfield X. (2004). Codon-optimized Gaussia Luciferase cDNA for Mammalian Gene expression in culture and in vivo, Molecular Therapy, 11. pp 435-443

Wiles S., Ferguson K., Stefanidou M., Young D., Robertson B. (2005). Alternative Luciferase for monitoring bacterial cells under adverse conditions, Applied and Environmental Microbiology, 71. pp 3427-3432

Reaction or catalyst? Which started life?

If only the question was as simple as ‘which came first the chicken or the egg’ (answer: the egg). The question here is which came first - DNA or proteins. as we all know DNA stores the code to make proteins but proteins are needed to read this code and make more DNA. Obviously you can’t have one without the other. So the RNA world hypothesis was born. RNA can do both store information and catalyse reactions, perhaps even catalyse its own replication. I believe this is a beautiful theory. However beauty is only skin deep. There are real problems with this explanation of the origins of life apart from the obvious difficulties in testing it, which is true for any explanation of the origin of life. RNA is famous for being chemically unstable. It can break itself down quite easily (especially in alkaline conditions). Also where did the RNA come from? I don’t mean did it come from outer space (because radiation levels in space are so high RNA or cells could not have survived). I mean how the molecules were created on earth. Like it or not RNA is a complex molecule and no experiment trying to recreate the primordial soup has ever found RNA nucleotides. Or any really complex molecules, only the simplest of amino acids have been made.

This has lead to another explanation for the origins of life. Perhaps thinking of life as a bunch of replicators has blinded use to it. Living organisms can also be thought of as chemical factories. Genes and their products are simply there to control these reactions. Some believe it was chemical reactions that came before the proteins or RNA that catalyse the reaction. This is called the metabolism first theory. People who follow this theory have described what is needed for a chemical system to be the beginnings of life. 1) A boundary or form of membrane is needed to keep life and non-life away from each other. The 2^nd law of thermodynamics states the universe my decrease in order but life increases in order so inside the boundary entropy decreases but this generates heat that causes an increase in entropy outside it. 2) An energy source must have existed. Perhaps some sort of redox reaction to power the chemical reactions in the metabolism first model. Radiation may have been used. 3) The energy source must be linked to the other chemical reaction. For me this is difficult to see how this could happen without proteins there to help things along but perhaps if I knew more chemistry it would be clearer. We use ATP as our energy currency but how could redox reactions or radiation be linked to these ancient chemical reactions? 4) The chemical reactions must be able to change and evolve. If a cycle of reactions was created where A became B and B became C and C became D and D became A again we have something to expand upon. If we had a carbon input such as E we could take compounds off the cycle and expand it (see diagram). These reactions could be powered by a redox reaction of X to Y. Eventually complex molecules could be created 5) One final requirement for these reactions to have been the origins of life is need and that is to be able to replicate. It is hard to imagine how this is possible before a lipid membrane existed for it to divide into two. If possible this would have allowed for Darwinian evolution through the competition for recourses.

The RNA-first approach has some support for it. Minerals have been found that contain boron in ‘containers’ or ‘bowls’ in Death Valley which could help create the ribose sugar in RNA. If such pores with boron existed billions of years ago RNA could have been created. Laboratory experiments have shown some randomly generated RNA molecules can catalyse the addition of an ATP molecule to itself. This is tested by using an ATP molecule with a sulphur atom not an oxygen atom and using a column that pulls out the sulphur containing RNA molecules only. RNA can carryout many reactions such as making and breaking DNA and RNA links and amide bonds and even make links with sugars. So their is diversity in RNA’s ability to catalyse reactions but its limit appears to be speed. It is possible one reason proteins took over from RNA as life’s catalyst because proteins are faster as well as because protein is more stable. Metabolism first has the great weakness of not having much lab experiments to support it but only computer simulations.

I believe these two theories could work together. Perhaps it is only because I find the rna world aesthetically pleasing i want to save it but these reactions could eventually become so complex they make rna. Over time these built up and started to take control and then made proteins to do much of there job when dna then took over as the info store and rna was simply the messenger and helps out in only a few reactions today. This nicely explains why the formation of the peptide bond in the ribosome is still done by rna. We could go even further into theory and suggest there was a polymer before RNA that could act as catalyse and self replicator. PNA has been suggested. Instead of having the sugar-phosphate backbone like RNA and DNA it has peptides attached to bases forming a backbone. Sadly such a molecule does not exist in our cells today or leave fossils in the ground for us to examine so we cannot test if PNA was really the first molecules that lead to life. If PNA did exist it all became RNA and then DNA.

At the moment we have many ideas about what may have been involved with the start of life on earth but it is difficult to prove anything. What we can do is explore the potentials of these molecules or chemical systems. After all theories about the origins of life cannot be tested directly but the do make predictions and by testing these predictions we can hopefully learn a lot.

References

Albert et al. (2002) Molecular biology of the cell. 4th edition.

Shapiro (2007) A simpler origin for life. Scientific American 296: 24-31.

The Lives Of Stars

I am currently reading Carl Sagan’s classic popular science book Cosmos, and felt inspired to write about a chapter I particularly enjoyed. Chapter IX, ‘The Lives of Stars’, takes us on a journey through space and time, looking at the Sun as well as some of its distant cousin stars, all of which behave in strange and wonderful ways.

Probably the most surprising thing about stars is that it all boils down to simple chemistry. Four hydrogen nuclei will combine, under very high gravitational pressure and temperature, to form a helium nucleus and emit light as a gamma ray photon. This is the almost disappointingly simple answer to a question that has tormented humankind since we first realized that there was actually a huge hot bright disc up there, a question that has led millions to invent all sorts of farfetched hypotheses and religions to explain it away. It really gets interesting when you travel backwards and forwards in time to see where it all comes from and where it will all end up. Let’s take it from the top, then.

The big bang was an explosion and rapid expansion of the fabric of spacetime, which consisted of some matter in the form of protons, neutrons and electrons, as well as a huge amount of nothing. The rapid cooling that followed due to this expansion caused these elementary particles to form hydrogen and helium gas clouds. The explosion itself was uneven, so clouds began to form clusters of various sizes, collapsing into themselves under the force of gravity. These massive clouds of gas are the birthplaces of millions of stars, eventually forming the galaxies we see and live in today, such as the Andromeda galaxy pictured on the right. Stars consist of that same gas having collapsed into itself at various points in space.

Stars are essentially massive engines that burn hydrogen. When temperatures in the core of a star are high enough (over 10 million degrees), the collapse stops as the outer layer is held back by the combustion taking place in the core. The photons emitted by the reaction take a million years to reach the outer layer. The sun has been a simultaneously exploding and collapsing hydrogen bomb for about 5 billion years, and it will continue to behave that way for about as long. Eventually, all engines run out of fuel, and so do all stars, but that does not always mean their death.

As the hydrogen runs out, the reaction will begin to cool and the star will expand outwards, engulfing the inner solar system. However, it will soon begin collapsing again under its own gravitational force, this time until temperatures get high enough to burn helium. Sagan compares this beautifully to a Phoenix rising out of its ashes, except this is not just an ancient myth but a real event that is constantly happening throughout the universe. The remaining hydrogen left over in the expanded region of the star will burn while helium burns at the core at higher temperatures. This is a red giant, with a hot carbon and oxygen-producing helium reactor in its core and a planet-engulfing hydrogen-burning outer region.

When the helium runs out, it does mean the end for most stars. A new expansion will take place, and the star will shoot out concentric shells of gas that will form the planetary nebula (pictured below). At this stage, the Sun would engulf Pluto. A few more massive stars can recollapse and burn carbon and oxygen for a while, but this is not very common. After the sun expands for the last time, the solar system will become a blue and red-fluorescent dead world. Billions of years later, the exposed core will become a white dwarf, and eventually a cold, dead black dwarf.

A planetary nebula

There are so many different aspects to this story that rival any storyteller’s wildest imagination. The poetic elegance of the lives of stars masks their terrible and devastating effect on the observing civilizations of their orbiting planets, but the universe is of course entirely indifferent and apathetic. I strongly recommend Cosmos to anyone who wants to catch a glimpse of the amazing things astronomy has discovered, especially since the invention of the radio telescope which can take us right to the edge of the universe.

Someone else in the universe already posted this but in a galaxy far far away

I would like to start off by saying I know nothing about physics apart from the excess of Sci-Fi I watch when I was younger and the random conversations I have with my physics friend Bob. Nevertheless I do like it despite it never making any sense. As a biologist there is a reason behind just about everything (ie evolution has shaped it all). It unsettles me when I don’t know why something is the way it is (so most of immunology then). Before reading the rest of this you need to assume a couple of things about the universe for what I am going to say to make sense, they are apparently correct but what do I know. Firstly space is infinite or extremely large (according to Prof Max Tegmark evidence for a small universe or donut shaped one is weak and he believes the universe is infinite) and secondly that matter is evenly spread out throughout space (not just clumped around us).

For the first part we will say our universe is the part of space we can see (the outer edges are determined by the age of the universe and how fast light it, as it gets older we can see more because more light has had time to travel to us). This is also called our hubble. Level I multiverse or parallel universes are simply hubbles out there that are like ours (and many more not like ours). If space is infinite all arrangements of matter that are possible exist! I like to think star wars and LOTRs obey the laws of physics so they could exist. Everything in level I has the same laws of physics as our universe because it is only an extension of ours. Out there, there are more hubbles just like ours but very far away. One estimate says one just like ours in every way could be 10 to 10¹¹⁸ metres away! Closer will probably be some hubbles that are very similar to ours but slightly different. Their will be an infinite number of all hubbles because space is infinite (from my understanding). I think these estimates are far to low and don’t take into account some things but it gives you an idea that even a low estimate is amazingly far away.

That was the easy stuff. Level II multiverse (or parallel universes) is less accepted than level I but still apparently explains a lot of things in physics. It helps biologists in their fight against pro intelligent design arguments. It is where many multiverses (same as in level I) exist but each multiverse is separate and has different physical constants or number of dimensions. This explains why our universe just so happens to be able to support life – it isn’t custom built to support life but is just one possibility of how the universe works. We have 3 spatial dimensions and one time dimension but if we had more time dimensions events would be completely unpredictable and if we had more spatial dimensions atoms would be unstable. If the mass of an proton was slightly larger it would decay to fast for molecules to be made so obviously nothing for evolution to act on and life to form. Each multiverse is still infinite within a sea of inflating infinite empty space (don’t ask me how you can have infinite space inside infinite space, because I just don’t understand). We can never travel between these multiverses even at the speed of light because they are moving away from each other faster! That sounds like science-fiction to me.

Level III is the one that interested me the most as a child (yes I have always been this way, Egon Spengler was my favourite Ghostbuster…need I say more). Every choice means you have to choose a path to follow. But in level III all outcomes exist but the other choices exist in another universe and not in space as we know it but ‘elsewhere’. However the choices I am talking about take place on the quantum scale! This goes back to Schrödinger’s cat (if you don’t know what it is, it would be best to look it up before continuing). From what I understand all it means is all states or positions exist until someone observes what state it is in. So something is both on and off or dead and alive until someone checks. This is called a superposition. An alternative theory is that both do exist but new universes are created to accommodate the other possibilities. It is like rolling a die that is only governed by the rules of the quantum world and not the overall rules of the universe. Because it is only on the quantum scale the outcome is completely random (from what I understand). According to level III multiverse it will not land on a 1 or a 2 or a 3… but will land on all six values at once. How; each one exists in a different universe, easy! And we thought biology was screwed up, at least it usually makes some sense. The outcome of level III is the same as for level I and II, more universes most slightly different from ours. The difference is how it is made.

Level IV is the one I feel least confidence about explaining. It is there to explain why our universe works under a set of specific mathematics and not controlled by other models. Universes that work using different mathematical models may exist outside our spacetime and work in completely different ways. They work using different laws of physics even more different to ours than by multiverses in level II. Level I, II and III were created by the same big bang. Level IV exists outside spacetime and will have had there own starting events but more level I and II (and III) could have been created by other big bangs as well. In level I and III you will have a Doppelgänger but in II and IV space will be so radically different you will not. Well I think you might if a parallel universe created in level II or IV is very similar and works off the same rules as ours.

All of these theories do make some predictions so they no longer lay in the realms of metaphysics but of real science. So in the next few years we may see some of these confirmed or rejected as fact. Whether they exist will have little impact on our lives. Will it comfort you to know someone out there in the infiniteness of space is in exactly the same situation as you or better of, or in a worse situation! I don’t think it will keep me awake at night. Nothing we can do will have an affect on them. I only discuss this because I find it interesting. But I find most things about our world its place in the universe interesting. We didn’t evolve to understand things like this but we did evolve to ask question about our environment.

For more information see Scientific American special report on Parallel Universe by Max Tegmark or his website:

http://space.mit.edu/home/tegmark/multiverse.html

this has more references.

Please ask me questions, no promises I can start to answer but I have thought about the topic for a while and would like to hear your thoughts and see of you have the same questions as me.

Colds and the cold

My mum always told me if I didn’t wear socks and shoes and had cold feet I would get a cold! Some evidence suggests that this old wives tale may be true. Adults have about 3-5 cold infections every year (unless you are a student and it is considerably more than this) and the symptoms are so well know and easily recognised that people self diagnose and there are no special tests doctors perform to say you have a cold, they go off these same old symptoms as the public. Saying what is a cold or flu is not easy but flu is much worse than most common colds, but a bad cold could be easily confused with a mild case of flu. Someone’s reaction to an upper respiratory infection depends more on the person (such as there stress level) than the virus that infects them. There are more than 200 serotypes of viruses (viruses with different antigens) that cause the common cold. Once infected by one of them you have antibodies to protect you from it causing another cold but there are plenty more waiting in the wings to hit you the next time exams are approaching. The rhinovirus is the most common cause of the common cold. The symptoms we experience when we get a cold are not the virus damaging us but our body reacting to the virus. Histamine triggers nerves in the noise to fire and tell the brain to make you sneeze and a sore throat is from a small peptide (bradykinin) signalling to nerves to tell you something is wrong. The colour of you lovely mucus changes from clear to yellow to green as more leukocytes (such as neutrophils) are recruited to fight the infection (Eccles, 2005). The majority of infected people are believed to not have any symptoms or only very mild ones. These are called cub-clinical infections and they can spread to others who will develop a full blown cold (Eccles, 2002).

The question is does the cooling of the body’s surfaces increase the chances of you getting a cold. The name cold suggests a link to me. The usual answer to why we get more colds in winter and cold weather is because we all crowd around in close spaces indoors and breath the same air. However I disagree with this. I do not change my habits during the winter and summer, I live in the same house with the same people who stay in the same no matter what the weather and go to school/uni and sit in the same classes with the same amount of people no matter what the weather. So how can you explain why I get more colds in the winter? I guess my mum is right. Because my feet aren’t warm enough…well there is now evidence to support my mum’s theory. Eccles and Johnson (2005) did find that when people put there feet into cold water and were exposed to a virus they were more likely to develop cold symptoms than those who did not have cold feet. But what are the mechanisms that mean cooling of the body’s extremities to let the virus get the upper hand? Vasoconstriction happens when you get cold, therefore less blood flows to the upper airways. This restricts the supply of heat and nutrients to leukocytes that eliminate viruses in a non-specific manor and reduces phagocytosis. Virus replication may also be increased, rhinoviruses replicate better at 33^oC than 37^oC. This could all cause a sub-clinical infection to become a full blow cold! Runny noise and all. Vasoconstriction helping cause a cold may also explain why some people get more colds than others. It has been shown that people who get more colds a year have a greater vasoconstriction response than those who only get a couple of colds per year (Eccles, 2002).

There are a lot of questions about colds and how they cause disease and how we catch them. But these diseases do not cause a lot of deaths, only reduce work output and the symptoms can be treated directly. I am just interested in what happens when I am ill.