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More on the Lac Operon

A while ago I wrote two posts about the Lac Operon here. The first pointed to an animation by McGraw Hill Publishers that did a pretty good job illustrating how the operon works. In the second post, I highlighted the notion of polycistronic messages (more than one gene per mRNA molecule) and how this allows for control of a number of related genes at once – a trait not shared by eukaryotic cells. In that second post, I also finished with a graph of how cells grow in the presence of glucose and lactose.

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Cell Growth in the presence of glucose + lactose – As glucose is depleted, cells adjust to lactose digestion

One feature of that graph (reproduced here) that is notable is a little bump in the growth rate as glucose runs out and the cell converts to lactose digestion. A second important feature is that the rate of growth slows when the cell is burning lactose as its primary fuel.

 

Together, these features suggest that the cell is regulating lactose digestion very closely. In fact, there are two primary mechanisms of this regulation to appreciate. The first is that the lactose-digesting enzymes are controlled together on an operon that is regulated by lactose itself (or at least we can assume so for simplicity’s sake). In the absence of lactose, no lactase enzymes are made and no lactose is used as fuel. The reason for this is obvious when you look at the slope of cell growth under glucose metabolism (left) and lactose metabolism (right). Clearly, growth is SLOWER when lactose is used as fuel.

Therefore, so long as there is glucose, it is pointless to digest lactose at the same time. So it is best to only turn on the lac operon in the ABSENCE of glucose – regardless of whether lactose is present of not.

If glucose is absent and lactose is absent, turning on lactase enzymes is still useless. However, slow growth is better than no growth. So we should have a mechanism to turn on the operon when there is lactose in the environment.

Here’s a matrix of ideal regulation:

Screen Shot 2014-03-31 at 2.18.05 PM

How can a little, mindless bacteria achieve this exquisite control?

Simple: By using two regulators. One for glucose and one for lactose. Only when both conditions (glucose-, lactose+) are met do we make lactase.

Structure of the Lac Operon

operon1

First, lactose itself serves as an inducer. In the absence of lactose, a regulator protein binds to a DNA site between the polymerase binding site (the promoter) and the structural genes (the enzymes). When the regulator binds, its presence physically prevents the progress of RNA Polymerase.

When lactose is present, it binds the repressor protein in a way that causes its shape to change in a way that can no longer bind the DNA. The repressor then drifts away from its binding site allowing RNA Polymerase a clear shot to the structural genes.

operon2

However, RNA Polymerase is not always parked on the promoter waiting for the repressor to be removed. Its binding requires another protein to help stabilize its interaction with the DNA. This second protein is the CAP protein. The Catabolite Activated Protein. However, CAP alone will not bind either. It requires a signaling molecule called cyclic AMP (cAMP). cAMP is readily broken down when glucose is in the cell, so it only accumulates when glucose is absent. In that case, cAMP accumulates and binds to the CAP protein, which then binds to the CAP site. This site is located adjacent to the promoter, but on the side away from the structural genes. When CAP binds, it assists in recruiting the RNA polymerase to the promoter.

operon3

Therefore, if only one condition is met, it is insufficient to promote gene transcription. Only when the CAP+ cAMP protein is bound will the Polymerase be recruited. And only when lactose is present, will the repressor protein let the Polymerase pass.

operon4

In terms of the matrix we set forth above, we can see that these molecular interactions result in exactly the regulation that is optimal:

post-operon

 

 
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Posted by on March 31, 2014 in Uncategorized

 

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Using Antibodies as vaccine delivery vehicles

Antibodies are glycoprotein molecules synthesized by plasma cells (mature, activated B cells) with the capacity of binding to any potential antigen epitope. For a review of lymphocytes and how they are activated, see this link where you will find more information about antibody production in response to ’challenge’.

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An (IgG) antibody with basic structural features labeled

Antibodies are the natural products of these plasma cells and function in a variety of ways to effect immunity. Most basically, they bind and may interrupt the function of the target molecules or trigger a response disadvantageous to the pathogen. In addition, a number of other functions are mediated by these molecules, including recruitment of complement and of phagocytic cells that will digest and inactivate the cell / antigen.

Therapies, such as vaccines, are designed to separate and eliminate the disease-causing elements of a pathogen from those that generate an immune response, thereby initiating a normal immune response to antigens without the dangerous exposure to live pathogens. Most often, these are prophylactic vaccines that initiate the development of immune ’memory’ prior to any disease exposure.

In some cases, therapeutic vaccines do much the same job, but are used to ’jump-start’ an immune response that has failed to initiate naturally for some reason (this may be because the target of the therapy is very similar to ’self’ as is the case with cancer), or because a long-term, chronic disease has fooled the body into tolerating an unwanted condition.

Additionally, some molecular therapies provide passive immunity by administering exogenous antibody that fulfills these functions. A weakness of these therapies is that, by providing pre-made antibody, potential antigens are blocked and no endogenous antibody response will be elicited.

A final use of antibodies, to be elaborated further here, is to provide targeted delivery of toxins to pathogens or infected cells or to deliver antigens to the immune system.

Purpose: to trigger / amplify immunity to an ongoing infection or disease

Considerations:

1. Target protein or cell – what cell and what protein on that cell should be targeted to elicit the desired immune response?

2. How to get antibody to the site where target cells are present?

3. What is the desired response / activity of the target cell?

4. What, if any, molecule is being delivered to these cells?

5. Lastly, how can efficacy be measured and what are the objective endpoints that will be used to determine whether therapy is effective?

Although this antibody is not currently in use therapeutically, I will use, as an example, one that I made while working for a biotech company some years ago.

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An antibody with an antigen conjugated to the Fc portion

The antibody we used specifically bound to the macrophage mannose receptor (MMR) expressed by macrophages and the similar phagocyte cells, dendritic cells. Natively, this protein binds to a sugar, mannose, that is commonly charged to protein molecules. Once bound, the MMR will direct receptor-mediated endocytosis of the bound protein and deliver it to endolysosomes for processing and presentation upon MHC class II molecules (see animation below). As explained in the link, processing and presentation lead to the activation of T Cells and the resulting immune response.

Using an antibody that targets this molecule (MMR), a target compound can be fused to the antibody (chemically or genetically) leading to the precise delivery of this compound into the cell and the generation of a response. The antibody will guide the (tumor) antigen to the phagocytic cell. In this way, the antibody serves only as a vehicle. This vehicle takes its passenger, the antigen that we would like to generate an immune response against, and inserts this antigen into the processing and presenting apparatus of these ‘professional’ antigen presenting cells.

Animation of Antibody delivering a Target Antigen to an APC:

 
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Posted by on December 26, 2013 in Uncategorized

 

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Cellular robotics? A cute video summarizing cellular functions from TedEd

Check out this video. I think I like it, but I’m not positive yet. It’s so well done that I’m kind of taken by the aesthetics, however, I’m not sure that this makes cell biology easier to understand. What’s your opinion?

 
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Posted by on November 25, 2013 in Uncategorized

 

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Mitosis Animation

I found this excellent animation of mitosis today and wanted to make it available here for anyone who wants to clarify their understanding.

 
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Posted by on October 15, 2013 in Uncategorized

 

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Nobel Prize in Medicine Awarded

Three scientists were awarded the 2013 Nobel Prize in Medicine to:

James E. Rothman, Randy W. Schekman 
and Thomas C. Südhof

for their discoveries of machinery regulating vesicle traffic, 
a major transport system in our cells

– from the press release from nobelprize.org

ImageThe full release can be found here. The  website  includes a link to a summary page illustrating the contributions of each scientist and how they come together to form a unified picture of vesicular traffic.

The New York Times article describing the award including an astute remark by NIH Director Francis Collins about the state of research in the United States. 

Dr. Francis Collins, the N.I.H. director, said in an interview on Monday. “Today we celebrate the three N.I.H.-supported Nobel Prize winners, but we’re being slammed by sequestration and a government shutdown.”

Even before the shutdown, scientists were facing severe budgetary difficulties that restrict the kind of research that led to this year’s Nobel Prize, Dr. Collins noted. “How many potential future Nobel Prize winners are struggling to find research support today, or have been sent home on furlough?” he said. “How many of them are wondering whether they should do something else — or move to another country? It is a bitter irony for the future of our nation’s health that N.I.H. is being hamstrung this way, just when the science is moving forward at an unprecedented pace.”

 
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Posted by on October 7, 2013 in Uncategorized

 

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A look into cell division

ImageIt’s that time in the general biology semester where we transfer our attention to cell division.  Having already discussed a number of basic principles like the laws of thermodynamics and a touch of chemistry, and cellular functions such as the flow of energy and the flow of information, it’s now time to look at how cells reproduce themselves.

In this chapter we should be recalling all the parts of the cell and accounting for how they get sorted into the developing ‘daughter cells’, and also recall the role of information, in the form of DNA, and how this is apportioned into the daughter. Of course we will spend most of our time focusing on the distribution of DNA, but we should always keep in mind what we know of other structures and organelles.

I previously wrote an essay describing cell division in humans that marries this information with the subject of the next unit, genetics and inheritance. You can find that text here. Therein, I briefly address one of the oddities of eukaryotic cells, the mitochondria. Mitochondria are odd because they live in our cells as strange symbiotes that share their energy with us in exchange for protection and a supply of nutrients. The theory describing this relationship was proposed by Lynn Margulis, and is widely accepted today. A description of her theory can be found here.

Because Mitochondria (and chloroplasts) are pseudo-autonomous cells, they must replicate themselves. A cartoon and some micrographs that illustrate this process have been borrowed from Nature Reviews.

ImageThe process involves an interaction with the Endoplasmic Reticulum, that guides an assembly of molecules that constrict around the Mitochondria eventually effecting its division into to smaller organelles. What this image does not include is the replication and separation of the mitochondria’s own circular DNA, a process that necessarily precedes the actual division of the organelle.

Altogether, there’s a lot to keep in mind when examining cell division. Why is this cell dividing? How are the instructions for life (DNA) being distributed between daughter cells? What does the daughter cell need in order to survive on its own? How do these parts / organelles handle their own division between the cells? And what would happen if any of this went wrong along the way?

 
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Posted by on October 7, 2013 in Education, Uncategorized

 

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Aside
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Substrate Level Phosphorylation

Several processes occur during normal eukaryotic metabolism to create ATP. During glycolysis (the breaking of sugar) both prokaryotes and eukaryotes use energy from the chemical bonds in the sugar to make ATP by directly transferring phosphates from the substrate molecule to ADP, resulting in ATP. Predictably, this process became known as ‘substrate-level phosphorylation. Both Cell Respiration, occurring in the mitochondria, and the light reactions of photosynthesis, occurring in the chloroplasts, also made ATP, however, no one understood how this occurred as no intermediate substrate molecule bearing the phosphates groups was known.

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1978 Nobel Prize in Chemistry winner

The Peter Mitchell, working at his own, privately funded research foundation, tackled this problem and determined that the power to make ATP came from two processes linked indirectly. For his work in this area, Mitchell won the 1978 Nobel Prize in Chemistry “for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory”.

Model diagram of electron transport and H+ translocation across the membrane

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Electron flow carries H+ across the membrane

Process#1: One of these processes is the electron transport chain (E.T.C.) during which a high-energy, excited electron is passed down a series of membrane proteins. As the electron is passed, it sometimes pulls hydrogen ions (H+) along and passes them across the membrane (see the cartoon illustration of this model by Mitchell). As a result, this process creates an electrochemical gradient across the membrane with more H+ on one side compared to very few on the other.

Process #2: As we know, these gradients will ‘want’ to resolve themselves and move towards equilibrium (by diffusion). There exists a special channel protein that H+ may pass through from the side of the membrane with a high concentration of these ions to the other.

“Each chemical species (for example, “water molecules”, “sodium ions”, “electrons”, etc.) has an electrochemical potential (a quantity with units of energy) at any given location, which represents how easy or difficult it is to add more of that species to that location. If possible, a species will move from areas with higher electrochemical potential to areas with lower electrochemical potential; in equilibrium, the electrochemical potential will be constant everywhere for each species”

                         -from the wiki page on electrochemical potential

I prefer to imagine the membrane and ions as a hydroelectric dam with water building up on one side and a relief passage through the dam.Image

Just as energy is captured when water rushes through the dam, H+ ions coming through the channel protein are used to power an enzymatic subunit that synthesizes ATP.

Sigma-Aldrich provides an excellent animation illustrating how ATP Synthase operates as both a H+ channel and an enzyme making ATP.

http://www.sigmaaldrich.com/life-science/metabolomics/learning-center/metabolic-pathways/atp-synthase/atp-animation.html

A conceptually simple set of experiments provides the evidence supporting this model. Here, an artificial membrane is made incorporating ATP synthase and bacteriorhodopsin. The rhodopsin molecule is capable of transporting H+s across the cell membrane when it is struck by light. Given sufficient supplies of H+ ions, ADP and Pi, ATP will be formed when a light source is present. In the absence of light, no H+ is transported and no ATP is made.

When a H+ carrier molecule that can diffuse through the membrane is introduced, this carrier maintains equal amounts of H+ on both sides of the membrane. Further, even when light is present, H+ is pumped across the membrane and then re diffuses back creating little or no ATP. This is illustrated in a cartoon from Albert’s Essential Cell Biology:

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Chemiosmosis defined experimentally

Chemiosmosis and the work of Peter Mitchell

 
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Posted by on October 5, 2013 in Uncategorized

 

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