Friday, 3 March 2017

Sweetness from light? Glucose is my molecule for March

My molecule for February was the metabolic, blood-sugar regulator Insulin, which made me realise that I haven't included glucose in my rogues' gallery of biologically important molecules. So I am putting that right for March with an overview of this molecule that has been central to the field of Biochemistry since it first began. The image on the left shows a cyclised molecule with 5 carbons and an oxygen atom at 12 o'clock. There are no double bonds, and importantly there are 5 hydroxyl groups at 2, 4, 6, 8 and 10 o'clock. However look closely, the OH at 10 o'clock is attached to a carbon atom that is not part of the hexameric ring. I think this is the easiest way to remember the molecular structure and chemical formula of glucose, which is C6-O6-H12 (for younger students, standard chemical structures exclude hydrogen atoms that are bonded to carbon atoms, to avoid clutter). This is sometimes called a Haworth projection. 

However, glucose is often depicted as a linear molecule (a Fischer projection, shown on the RHS) , with an aldehyde function at one end. The non-cyclic form of glucose is only present at low amounts (maybe 0.25%, in vivo: the remainder is cyclised). We are not done yet, since these are flat projections, and if there is one thing you will have realised by now, Biology is all about 3D shapes.  
In 3D terms, glucose molecules, when cyclised exist in a form often referred to as a "chair". This is shown on the left. The OH and H atoms that project from the ring carbons, do so in one of two ways: horizontally (equatorial) or perpendicular (axial). These two conformations exist in equilibrium, which can be of great importance when glucose encounters the active site of an enzyme. Can you think why? The other feature of glucose that again becomes important when it either acts as a substrate, or is involved in forming a linkage to a second sugar molecule: the orientation of the OH group at carbon 1. You can probably work out that the union of glucose and fructose (two monosaccharides) produces the disaccharide, sucrose, and it is the green OH at position 1, that forms a glycosidic bond to the OH at position 2 of a fructose molecule, which gives us α-D-glucopyranosyl β-D fructofuranoside, as shown on the right. The polymerisation of sugars, in particular glucose, produces carbohydrates such as starch and glycogen, both of which are found as storage products in living organisms, is a consequence of a dehydration reaction, which can occur at several OH groups. To recap then, the major form of metabolic glucose in living organisms, is cyclic and, since it is known to rotate plane polarised light in a right handed manner; we refer to it as a dextrose sugar (dexter is Latin for right handed). Which now brings me on to the question of why is glucose such a good source of energy?

Plants capture sunlight energy and infuse it with carbon dioxide to make glucose (among other things). As with any chemical reaction that occurs in vitro or in vivo, energy is involved. Just think of a typical petrol car. The fuel, gasoline is ignited in order to release the energy trapped in the chemical bonds within the fuel molecules to power the engine. In living organisms, sugars like glucose are placed at the start of an enzymatic conveyor belt and the trapped energy captured in the form of ATP , in order to provide us with the fuel we need to grow, move and think etc. The amount of energy contained in glucose is around 16kJ per gram (or if you prefer 3.75 kilocalories). If you consider the activity of a typical adult in the course of a day requires around 4 300 joules  (or 1000 calories), you can begin to appreciate the value of glucose as an energy source. I always think of bacterial media when I am trying to relate glucose quantities with living cells. Let's work it out. The molecular weight of glucose is about 180 and typically media contain 1% (w/v) glucose (10g/L). In my experience (depending on the strain and the growth conditions of course), a one litre culture of E.coli will yield around 5-10g wet weight of cells. Let's keep things simple, around 10g of glucose gives rise to 10g wet weight of bacterial cells (from a very small inoculum). Apart from a number of minerals, many lab strains of E.coli can grow on a very simple medium, provided with a carbon and nitrogen source. All of this is achieved by the catabolism of glucose which fuels, even in the case of a simple prokaryote, an incredibly sophisticated "molecular machine" that is capable of growth followed by cell division. It is clear I think that glucose is of paramount importance in most living organisms, justifying its choice as a molecule of the month, even if it is a late arrival!

Saturday, 28 January 2017

Monika Madej and Alanya Roberts - Green Flouresecent Protein (GFP)

I have really enjoyed Project Based with my teacher Dr. Dyer. My group has moved onto our choice module which was Microbiology and Biotechnology. Our project consisted of using a recombinant strain of E.coli that is able to produce Green Fluorescent Protein (GFP) because it contains the GFP gene which originally came form a  jellyfish called Aqueora Victoria. I thought this module was very interesting and I could develop my microbiology skills further.  I think the laboratory lessons are a really good opportunity for me because I want to pursue my career in a laboratory scientific field, therefore thanks to those P.B. lessons I am have started to develop skills which could be used in the future at university or in a job. This will be a massive advantage for me compared to other students in other schools.

Monika Madej

We used Green Fluorescent Protein (GFP), naturally occurring in Aequorea Victoria jellyfish but here produced by a recombinant strain of E.coli. This protein has been used by Scientists to identify molecules under a light microscope and is particularly useful in identifying drug targets. Hornby (2014) says that luminescence is the emission of light without the input of heat which is important in understanding fluorescence. After starting our culture, we investigated the effects of temperature on the growth rates of E.Coli GFP. We found that E.Coli GFP grew at a faster rate at the temperature of 25 degrees celsius over 13 hours than at 37 degrees celsius. This is surprising as the optimal temperature for E.coli growth is around 37 degrees celsius.

Alanya Roberts

Friday, 27 January 2017

Insulin: Molecule of the Month for February 2017

I always wrestle with my choice of molecule a few days before my Google Calendar sends me an irritating, but essential reminder to start writing my Molecule of the Month entry. I often set my sights on one molecule and something at the last minute tells me to change tack. This time, I was contemplating an influenza post, but then I read an article about Dorothy Hodgkin (shown left), and realised I have never written about insulin. So, to put this right, here then is my homage to the late, great crystallographer, Dorothy Hodgkin, or rather the molecule that she made her own.
With Roy Orbison's classic "Pretty Woman" topping the charts, the Nobel Committee of 1964, announced that Dorothy Crowfoot Hodgkin had been chosen to receive the Nobel Prize in Chemistry, for her work in the field of X-ray crystallography, specifically, the determination of the molecular structure of  Vitamin B12, which at the time represented a major intellectual and technical achievement in the field (RHS). It wasn't until around five years later that the structure of insulin would emerge from Dorothy Hodgkin's laboratory, nearly 35 years after she carried out her first experiments on a molecule that captured her imagination owing to insulin's extraordinary impact on human physiology. Before I launch into the molecule itself, it is worth pointing out that having won the Nobel Prize in 1964, Dorothy Hodgkin received the Order of Merit a year later; only the second woman recipient at the time: the first being Florence Nightingale! However, I do like the fact that alongside her scientific prizes, she was awarded the Lenin Peace Prize. In fact she was awarded the Peace Prize in 1987, just before she opened the Krebs Institute at Sheffield. Since the Prize was discontinued three years later, as the former Soviet Union experienced Perestroika and Glasnost, no other Scientists will ever receive it!

Most of us knows someone who suffers from one of the two forms (Types I or II) of diabetes [a strange word that is derived from the Greek for a syphon: a device that draws off fluid]. Type I diabetes melitus, is an inherited disorder in which the insulin producing, pancreatic beta cells are compromised by an auto-immune attack, leading to a catastrophic loss of insulin and a classically "sweet" tasting blood/urine (hence melitus). Type II diabetes melitus (the melitus is often dropped), arises because the insulin responsive cells have become insulin resistant: so once again the blood is glucose rich, but insulin is still produced. Around 5-8% of the population (1 in 15 adults) have diabetes. However, only 0.5% of these individuals have the Type I form (who require regular insulin top-ups): the remainder generally develop Type II diabetes over the age of 40, and can keep the symptoms at bay by a change of lifestyle (a better diet and more exercise). Thanks to @WillSampsonUW, for providing this nice link, after reading the post.

So what doe insulin "look like" and how did insulin become one of the earliest protein based pharmaceuticals? In fact it was the first synthetic protein to be developed via both Chemistry (in the 1960s) and Biotechnology (in the late 1970s). Human insulin is a relatively small protein, comprising two chains (A and B) each containing 51 amino acids. The chains are held together by disulphide bonds. Insulin is found in all animals and is a highly conserved protein. Until the 1980s, Type I diabetes patients were routinely provided with porcine insulin to manage the symptoms of hyperglycaemia. The early "pharmacological" work of Banting, Macleod, Best and Collip (the first two received the Nobel Prize in Physiology or Medicine in 1923, and famously shared good fortune with the latter two!), identified insulin as the molecular solution to alleviating the symptoms of Type I diabetes. The drug company Eli Lilley began manufacturing, and clinicians administering, porcine insulin in the 1920s/'30s. In 1950, Novo Nordisk began manufacturing a longer lasting form of insulin: the porcine product continued to be used until around 1980, when recombinant insulin became the main form, developed by Genentech in San Francisco using recombinant DNA technology.

I can't possibly leave out Fred Sanger (a role model for many Biochemists of my own and the previous generation of Biochemists) from the insulin story. The primary structure of insulin was determined by Fred Sanger in the early 1950s (earning him his first of two Nobel Prizes: see LHS). This pioneering work led to the determination of the sequences of many subsequent protein structures, which in itself paved the way for our understanding of protein evolution (which was subsequently accelerated by the work, that earned Fred Sanger his second, and perhaps better know Nobel Prize, for developing the principles of nucleic acid sequencing). I include Sanger's work for another reason. The sequences of human and porcine insulin differ by just one amino acid. However, Eli Lilley were producing a protein for injection into humans without any knowledge of its relationship to the endogenous human insulin, missing from diabetics. Today, this would not pass the drug regulatory authorities. However, like the use of penicillin, these ground-breaking biologics (as they are now often called) helped save many thousands of lives and it may be worth discussing this alongside thalidomide in a tutorial on science and ethics.

Returning to the structure of insulin, the human form (often referred to as Lispro) is shown on the right, with the characteristic Zn atom coordinated at the core of the molecule. The circulating form of insulin is generally a hexamer (as shown), but the active species is a monomer, which interacts with the Insulin Receptor, on liver cells, fat cells, skeletal muscle cells etc, to stimulate the assimilation of glucose into glycogen. The converse reaction, glucose release (or glycogen mobilisation) is induced by the yin to insulin's yang, glucagon, which is released from pancreatic alpha cells. The insulin receptor is a membrane bound, Tyrosine Kinase class molecule, which, in response to insulin binding, triggers a sequence of events that leads ultimately to the stimulation of the gluconeogenic pathway, thereby depleting levels of blood glucose. As I mentioned above; when glucose levels are balanced, the release of glucose is stimulated by glucagon. A general scheme is shown below (taken from the nice wiki site on the insulin receptor.

My final comments relate to the conservation of insulin's primary structure, which has emerged from the many animal genome sequences deposited over the last 15 years.The relationship between structure (primary begets tertiary, remember) and function, is a well established concept in chemistry and biochemistry, and I don't wish to suggest otherwise. However, as those of you who have read any of my earlier posts on the ramifications of Anfinsen's law and recent studies on structural equilibria among certain disease related proteins, suggests that primary structure-function relationships are a little more complex than we thought. The logic behind the highly conserved nature of the insulin molecule makes sense and, although this was not a pre-requisite (see above) for the early clinical use of the closely related porcine hormone, it is clear that insulin has many key interactions to make, for such a small protein. This raises an interesting tutorial point: is there any correlation between evolutionary conservation of primary structure and the molecular weight of the protein molecule in question? I hope you agree that insulin is a fascinating molecule, which has been at the heart of Physiology, Biochemistry, Structural Biology, Genetics and disease for over 100 years, and I have no doubt there is more to come...

Tuesday, 17 January 2017

Olivia Chadwick - The Whey to a Larger Mealworm

For my independent investigation i decided to work with the mealworms. I chose to investigate whether whey protein powder would have any effect on the rate of the mealworms growth and if it made them grow any larger than they would have if they had just been eating the porridge oats that they were usually fed.

To do this i put three mealworms in isolation and each day would feed one its usual diet of porridge oats, one whey protein powder and the other a mixture of the two. After a week of them being on these diets i weighed them. As i had expected the mealworm that was fed just the protein powder increased in weight the most however the other two mealworms lost weight, something i wasn't expecting to happen. If i were to do this investigation again i would monitor the mealworms more closely and i would also have more meal worms on each diet and possibly for a longer time as i don't think a week was long enough.

Overall i really enjoyed doing this investigation as it allowed me to increase my knowledge about all of the model organisms at UTC which i have found really interesting.

Friday, 13 January 2017

Tom Buchanan – Year 12

I have very much enjoyed working in the labs so far, I have had the opportunity to work with equipment I would otherwise have never had the chance to use. Such as the spectrophotometer which I have used to measure the absorbance of solution that I have produced in the lab. I enjoyed my microbiology module in which I produced GFP, a protein with the ability to fluoresce under UV light. Perhaps my most favourite so far has been during my enrichment activities were I have had the chance to work with the schools Daphnia Magna, this tiny, interesting organism is visible by the naked eye, however under microscope we are able to examine its transparent body and organs. I have conducted multiple experiments with them, such as monitoring how they respond to environmental changes like pH of their water, it is interesting to see how their new born respond to this change as I have discovered myself that they seem to produce more young when they are exposed to such a big environment change. I could probably go on forever about it. I have learnt key lab skills that are preparing me for a future working in a lab that I hope to one day have. In conclusion, my time so far in the labs has been fantastic, every day is different which is what makes it so exciting. I feel so lucky to be able to have these facilities available to me.

Daphnia on Drugs – Sana Nagi and Muna Ali

Our experiment was to see whether difference substances would affect the heart rate of Daphnia. The resting heart rate of Daphnia was recorded and they were then placed into ethanol, caffeine or water for 10 minutes and recorded again. We found that as predicted the ethanol slowed the heart rate, caffeine increased the heart rate and the water didn’t affect the heart rate as much. (As shown in the graph below).

This experiment gave us extra knowledge on the anatomy and physiology of the daphnia. It was very interesting to see how their body reacts with different substances such as ethanol and caffeine. We came across offspring developing inside the female daphnia which we found really fascinating. The fact that their bodies are opaque make them a great lab organism. During this experiment we had to repeat the steps 10 times for each substances which became really repetitive and frustrating. Another difficulty we faced was when we were counting the heart rate, as it was too hard to watch and record through the microscope at the same time and this caused anomalies. Overall the experience taught us a lot and helped us gain more experience for the future. 

Y12 Students share their experiences in the innovation labs

During the next few weeks I will publish a series of blog posts written by our year 12 students reflecting on the first term of Project Based Learning at the UTC. It has been a really exciting first term with the students taking part in a range of interesting projects in the labs.