Arsenic and Selenium

You should listen to this podcast from Quirks and Quarks about how University of Calgary scientist Judit Smits is trying to use selenium rich lentils from Saskatchewan, Canada to treat arsenic poisoning in Bangladesh. Well water in parts of rural Bangladesh have high levels of natural arsenic and this is a major health problem. Professor Smits, who is actually in the department of veterinary medicine, has done work using arsenic to treat selenium poisoning in animals. It turns out that arsenic and selenium, both of which can be toxic in high doses, effectively neutralize each other. They each seem to increase excretion of the other into the bile. So she hypothesized that selenium might counter arsenic poisoning but the interaction is nontrivial so it is not a certainty that it would work. Dr. Smits organized a study to transport ten tons of lentils from Canada to Bangladesh this past summer to test the hypothesis and you can hear about the trials and tribulations of getting the study done. The results are not yet in but I think this is a perfect example of how cleverness combined with determination can make a real difference. This study is funded entirely from Canadian sources but it sounds like something the Gates and Clinton foundations could be interested in.

2016-9-26. Corrected a typo, changed Saskatchewan to Bangladesh

New paper on steroid-regulated gene expression

I am extremely pleased that the third leg of our theory on steroid-regulated gene expression is finally published.

Theory of partial agonist activity of steroid hormones
Abstract: The different amounts of residual partial agonist activity (PAA) of antisteroids under assorted conditions have long been useful in clinical applications but remain largely unexplained. Not only does a given antagonist often afford unequal induction for multiple genes in the same cell but also the activity of the same antisteroid with the same gene changes with variations in concentration of numerous cofactors. Using glucocorticoid receptors as a model system, we have recently succeeded in constructing from first principles a theory that accurately describes how cofactors can modulate the ability of agonist steroids to regulate both gene induction and gene repression. We now extend this framework to the actions of antisteroids in gene induction. The theory shows why changes in PAA cannot be explained simply by differences in ligand affinity for receptor and requires action at a second step or site in the overall sequence of reactions. The theory also provides a method for locating the position of this second site, relative to a concentration limited step (CLS), which is a previously identified step in glucocorticoid-regulated transactivation that always occurs at the same position in the overall sequence of events of gene induction. Finally, the theory predicts that classes of antagonist ligands may be grouped on the basis of their maximal PAA with excess added cofactor and that the members of each class differ by how they act at the same step in the overall gene induction process. Thus, this theory now makes it possible to predict how different cofactors modulate antisteroid PAA, which should be invaluable in developing more selective antagonists.

Steroids are crucial hormones in the body, which are involved in development and homeostasis. They regulate gene expression by first binding to nuclear receptors that freely float in the cytosol. The receptor-steroid complex is activated somehow and transported to the nucleus, where it binds to a hormone response element and initiates transcription. Steroids can either induce or repress genes in a dose dependent way and the dose-response function is generally a linear-fractional function. In our work, we modeled the whole sequence of events as a complex-building biochemical reaction sequence and showed that a linear-fractional dose response could only arise under some specific but biophysically plausible conditions. See herehere, and here for more background.

Given the importance of steroids and hormones, several important drugs target these receptors. They include tamoxifen and raloxifene, and RU486. These drugs are partial agonists in that bind to nuclear receptors and either, block, reduce, or even increase gene expression. However, it was not really known how partial agonists or antagonists work. In this paper, we show that they work by altering the affinity of some reaction downstream of receptor-ligand binding and thus they can do this in a gene specific way. We show that the activity of a given partial agonist can be reversed by some other downstream transcription factor provided it act after this reaction. The theory also explains why receptor-ligand binding affinity has no affect on the partial agonist activity. The theory makes specific predictions on the mechanisms of partial agonists based on how the maximal activity and the EC50 of the dose response change as you add various transcription factors.

The big problem with these drugs is that nuclear receptors act all over the body and thus the possibility of side effects is high. I think our theory could be used as a guide for developing new drugs or combinations of drugs that can target specific genes and reduce side effects.

New paper on steroid-regulated gene expression

Recent paper in Molecular Endocrinology 7:1194-206. doi: 10.1210/me.2014-1069:

Research Resource: Modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay

John A. Blackford, Jr., Kyle R. Brimacombe, Edward J. Dougherty , Madhumita Pradhan, Min Shen, Zhuyin Li, Douglas S. Auld, Carson C. Chow, Christopher P. Austin, and S. Stoney Simons, Jr.

Abstract: Glucocorticoid steroids affect almost every tissue-type and thus are widely used to treat a variety of human pathologies. However, the severity of numerous side-effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (Amax) and EC50, or the position of the dexamethasone dose-response curve. Upon screening 1280 chemicals, ten with the greatest change in the absolute value of Amax or EC50 were selected for further examination. Qualitatively identical behaviors for 60 –90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the ten chemicals in a recently described competition assay determined their kinetically-defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

Did microbes cause the Great Dying?

In one of my very first posts almost a decade ago, I wrote about the end-Permian extinction 250 million years ago, which was the greatest mass extinction thus far. In that post I covered research that had ruled out an asteroid impact and found evidence of global warming, possibly due to volcanos, as a cause. Now, a recent paper in PNAS proposes that a horizontal gene transfer event from bacteria to archaea may have been the main cause for the increase of methane and CO2. This paper is one of the best papers I have read in a long time, combining geological field work, mathematical modeling, biochemistry, metabolism, and evolutionary phylogenetic analysis to make a compelling argument for their hypothesis.

Their case hinges on several pieces of evidence. The first comes from well-dated carbon isotopic records from China.  The data shows a steep plunge in the isotopic ratio (i.e ratio between the less abundant but heavier carbon 13 and the lighter more abundant carbon 12) in the inorganic carbonate reservoir with a moderate increase in the organic reservoir. In the earth’s carbon cycle, the organic reservoir comes from the conversion of atmospheric CO2 into carbohydrates via photosynthesis, which prefers carbon 12 to carbon 13. Organic carbon is returned to inorganic form through oxidation by animals eating photosynthetic organisms or by the burning of stored carbon like trees or coal. A steep drop in the isotopic ratio means that there was an extra surge of carbon 12 into the inorganic reservoir. Using a mathematical model, the authors show that in order to explain the steep drop, the inorganic reservoir must have grown superexponentially (faster than exponential). This requires some runaway positive feedback loop that is difficult to explain by geological processes such as volcanic activity, but is something that life is really good at.

The increased methane would have been oxidized to CO2 by other microbes, which would have lowered the oxygen concentration. This would allow for more efficient fermentation and thus more acetate fuel for the archaea to make more methane. The authors showed in another simple mathematical model how this positive feedback loop could lead to superexponential growth. Methane and CO2 are both greenhouse gases and their increase would have caused significant global warming. Anaerobic methane oxidation could also lead to the release of poisonous hydrogen sulfide.

They then considered what microbe could have been responsible. They realized that during the late Permian, a lot of organic material was being deposited in the sediment. The organic reservoir (i.e. fossil fuels, methane hydrates, soil organic matter, peat, etc) was much larger back then than today, as if someone or something used it up at some point. One of the end products of fermentation of this matter would be acetate and that is something archaea like to eat and convert to methane. There are two types of archaea that can do this and one is much more efficient than the other at high acetate concentrations. This increased efficiency was also shown recently to have arisen by a horizontal gene transfer event from a bacterium. A phylogenetic analysis of all known archaea showed that the progenitor of the efficient methanogenic one likely arose 250 million years ago.

The final piece of evidence is that the archaea need nickel to make methane. The authors then looked at the nickel concentrations in their Chinese geological samples and found a sharp increase in nickel immediately before the steep drop in the isotopic ratio. They postulate that the source of the nickel was the massive Siberian volcano eruptions at that time (and previously proposed as the cause of the increased methane and CO2).

This scenario required the unlikely coincidence of several events –  lots of excess organic fuel, low oxygen (and sulfate), increased nickel, and a horizontal gene transfer event. If any of these were missing, the Great Dying may not have taken place. However, given that there have been only 5 mass extinctions, although we may be currently inducing the 6th, low probability events may be required for such calamitous events. This paper should also give us some pause about introducing genetically modified organisms into the environment. While most will probably be harmless, you never know when one will be the match that lights the fire.



Michaelis-Menten kinetics

This year is the one hundred anniversary of the Michaelis-Menten equation, which was published in 1913 by German born biochemist Leonor Michaelis and Canadian physician Maud Menten. Menten was one of the first women to obtain a medical degree in Canada and travelled to Berlin to work with Michaelis because women were forbidden from doing research in Canada. After spending a few years in Europe she returned to the US to obtain a PhD from the University of Chicago and spent most of her career at the University of Pittsburgh. Michaelis also eventually moved to the US and had positions at Johns Hopkins University and the Rockefeller University.

The Michaelis-Menten equation is one of the first applications of mathematics to biochemistry and perhaps the most important. These days people, including myself, throw the term Michaelis-Menten around to generally mean any function of the form

f(x)= \frac {Vx}{K+x}

although its original derivation was to specify the rate of an enzymatic reaction.  In 1903, it had been discovered that enzymes, which catalyze reactions, work by binding to a substrate. Michaelis took up this line of research and Menten joined him. They focused on the enzyme invertase, which catalyzes the breaking down (i.e. hydrolysis) of the substrate sucrose (i.e. table sugar) into the simple sugars fructose and glucose. They modelled this reaction as

E + S \overset{k_f}{\underset{k_r}{\rightleftharpoons}} ES \overset{k_c}{\rightarrow }E +P

where the enzyme E binds to a substrate S to form a complex ES which releases the enzyme and forms a product P. The goal is to calculate the rate of the appearance of P.

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