What Does Hallatosis Smell Like

When the art technology Michaelis-Menten killed

start with the end, let us dead something that falls into disuse. Well this man, who in reality were two gentlemen not only are dead for many years, but here I am talking about his legacy. For those initiated into the life sciences, the Michaelis-Menten enzyme kinetics are not unknown, and most of clairvoyance to remember and why they wore, what we remember is that they were a nightmare, both to understand in the classroom like to make in the laboratory. For the uninitiated, is basically a way to study the characteristics of enzymes , which are nothing more than proteins that can facilitate a chemical reaction.

This type of kinetics was developed in the early twentieth century by Dr. Leonor Michaelis and Dr. Maud Menten , and is, at least for me, the paradigm of testing in vitro . I will not go in their complexity and mathematical assumptions. Their experimental procedure and roughly summarized: put an enzyme into a pot along with everything needed to make it work and at one point was added the substrate that the enzyme has to modify (phosphorylate, cut, break, destroy, ubiquitin etc etc. ..) and start timing. Note, that prior to this we have: purify a stable enzyme functional and invented a method to measure substrate disappearance or product appearance and standardize on a host of conditions that may affect the enzyme (ionic strength, pH, temperature, substrate concentration ...). This approach (as amended) has been used for almost 100 years to measure the kinetic constants (characteristics) of all known enzymes and purify them likely to be active in a boat. And it has been of tremendous use from the science of knowing in learning to pharmacodynamics (which saves lives). But the laser was the "in vitro " is coming to an end (let me use part because by all in the title).

Indeed in vitro experiments are still far from disappearing, but new technologies, most of those who are dedicated to studying the mechanisms of life, we are changing the boat in the living cell. Advances in genetic engineering, along with the discovery and development of new biological tools such as proteins and fluorescent substrates and very powerful microscopes (in addition to its highly refined optics with computerized control systems that allow the manipulation of micron level) do may be almost easier to follow the road map intracellular your "beloved protein X" to make a simple mix in a jar. You lose total control of all parameters of the reaction (in vitro fails ) but you gain, among other things, perceptions of what happens in "real life" also allows the analysis and quantification of processes as intracellular protein movement, or simple protein interactions in native environments (ie natural), that in vitro are almost impossible to discern.

green fluorescent protein (GFP image above), it absorbs blue-violet light and emits green light. Was obtained in the first version of the jellyfish Aequoria victory (abaj0). Successive improvements in genetic engineering have generated more stable types of GFP and bright, they are more useful.

This technology opens the closure of the study on single cell protein, is going to take a boat ( in vitro) data which reflect the overall statistical behavior of a population of millions of molecules, proteins or thousands of cells, to study tens or hundreds of molecules in a dozen or twenty of cells, but in depth. Here we realize that all behave in similar but not identical, which gives much more information. At the restaurant as an example the New York marathon. Speed \u200b\u200bstudy the long distance race of human beings. In an in vitro classic, taking into account all the people, collectively viewed from afar, who ran the last marathon (45 334), conclude that the speed of human being, 40km, is around 10km / h. But using this new cutting edge technology we are able to adjust much more and we can study the runners one by one, 20 of them for example to see that some are at 19 km / h, more than 10 km / h and other 7km / h. But let implausible examples and see some real examples:

fluorescence microscopy: That will be the base. In short, what is done is to attach a particular protein (X) to an existing fluorescent proteins such as GFP (green emitting) or Cherry (emitting in red). X (-GFP, for example) is expressed in the cell, the cell is put under the microscope, and hopefully that will be part of the cell is located our protein X (now green or red). And even if the protein moves, you can follow their movement over time (fluorescence microscopy in living cell).

Scheme how it makes a protein X either a fluorescent mark in this case GFP ( image )

real example fluorescence microscopy. Different cell types (rat skin, carcinoma ...), where the same protein is marked with GFP ( image )

FLIP: This acronym abbreviates the definition in English (I will not use it Images are available). This technique is called something like "fluorescence loss in photobleaching. Here is what is done as in the fluorescence microscopy but also living cell, using a laser is extremely precise "char" (photobleaching) continuously fluorescent protein together with our protein X in a small part of the structure are fluorescent staining. If the progress of time, the fluorescent signal is lost outside the area of \u200b\u200bphotobleaching, this means that our protein X blithely moves from one side to another, and when placed under the sun ... I mean, Laser tarnish. In short, moving the structure to make, and is not static.

FLIP and FRAP diagram, the rectangle indicates the area Cell to photobleaching (in English, photobleach) in each case ( Jennifer Lippincott-Schwartz and George H. Patterson, 2003, "Development and Use of Fluorescent Protein Markers in Living Cells. "Science )

real example of FLIP (now the image is in black and white because you have to pay to publish less, and in this case information is the same.) The square indicates the area to be "char" (photobleaching) continuously. In the second picture starts photobleaching Sool in the rectangular area, you can see how the fluorescence gradually disappears from the rest of the cell. (Jennifer Lippincott-Schwartz, Erik Snapp and Anne Kenworthy, 2001. Studying protein dynamics in living cells. Nature)

FRAP: "fluorescence recovery after photobleaching. It is like FLIP it in reverse. "Burn" the fluorescent protein in an area and waiting to see how (yes or no) fluorescence recovers.

real example of FRAP. The square indicates the area to be "char" (photobleaching) but in this case only for a short period of time. The three photos above are for a mobile protein, for 5 min. after the "charred", the area in question again becomes fluorescent. The three images below are a control, doing the same procedure in a protein that is not mobile, we can see after photobleaching the fluorescence remains square area. (Jennifer Lippincott-Schwartz, Erik Snapp and Anne Kenworthy, 2001. Studying protein dynamics in living cells. Nature)

FRET: "induced fluorescence energy transfer." The theoretical implications of this technique are more complex physically, but the idea is simple. A molecule is fluorescent light because it contains only one type (eg blue, in the case of GFP) and then the other issues but with less power (in this case green). Well, what happens if we use GFP with another protein that emits green light and reflects red (eg CHERRY), for if they are sufficiently close to each other, GFP can move to green energy and this issue CHERRY red. So if we want to know whether two proteins (X and Y) interact in their natural environment, joined to a GFP X and Y to Cherry and get into a cell. As in fluorescence microscopy, we will be able to see the location of each one separately if you use the "activating light" concrete of each. But if we use the green light to activate GFP and what we see is red (or yellow = red + green) shows that out of our protein indicates that our protein X and Y are very close (as Batablanca and Malina at some point) ... interacting (and not be wrong-headed).

Example FRET. This is another approach of this technique can observe how the shape of a protein under certain conditions (such as shortening the length). A, control. B, a protein fragment is bent causing the fluorescent protein approach. C, if the fragment is shorter, there is more efficiency in energy transfer between GFP and Cherry ( David Lleras, Samuel Swift, Angus I. Lamond, 2007. Detecting Protein-Protein Interactions In Vivo with using FRET Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM). Current protocols )

is true that after reading this tirade about these super-technological techniques, which are very impressive, one concludes that appear not serve nothing but to entertain a heap of scientists fascinated by the bright colors. However, their application, along with a host of other techniques (more or less leading and finicky), is enabling a deep understanding of the mechanisms of life. And this is important for many things, but two are key from my point of view. The first is that the mere knowledge (if only to know how is a bacteria or yeast ) is not a waste of time, although today not yield economic benefits or not know what it does or will. These techniques (and other, non-microscopic) is rooted in scientific and technical developments initiated 20 or 30 years ago (when they were only to be done), today are the tip of the spear which aims to eradicate Alzheimer's, cancer, AIDS, hepatitis, diabetes, malaria and a long list of pests that plague our world today. And the second is that all this search-application of knowledge is, at least from publicly funded research (at least in the 10 laboratories that have stepped in my life), applying a level of caution and environmental and personal protection that if you take into other jobs or everyday life, the most we had not cut with a knife in our lives.