My two reflective pieces for my published papers

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Topic 1 Reflection: “Neurological Effects of Caffeine”

Reference:

“Neurological Effects of Caffeine,” Medscape, accessed on March 29, 2013, http://emedicine.medscape.com/article/1182710- overview#aw2aab6b4

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Thought to be a drug addiction, caffeine got the “award” for being the most consumed psychoactive essence. Caffeine lead to various neurological effects on the body. Related to uric acid in design, 1, 3, 7-trimethylxnthine (caffeine) undergoes “oxidation and demethylation” when metabolized. Derivatives such as acetylated uracil, 1-methyluric acid and 1-methylxanthine are urinary metabolites which occurred from 1, 7-dimethylxanthine. However the conversion of methyl xanthine to uric acid has not been evidentially supported. Genetic and the environment played a role in the determination of the rate at which methyl xanthine was eliminated. Metabolism of methyl xanthine followed “first order kinetics” but at higher concentrations, obeyed “zero-order kinetics” due to metabolic enzymes being saturated. The presence of other disease in the body also played a role in the breakdown of methyl xanthine. With a half-life of 3-7 hrs in plasma, methyl xanthine’s life increased through late pregnancy and ‘long term use” of “contraceptive steroids.” Methyl xanthine, “Translocated intracellular calcium,” “increased the build-up of cyclic nucleotides” and “Blocked adenosine receptors.” Caffeine interfered with the uptake and cache of calcium through the ‘sarcoplasmic reticulum” in striated muscles whilst it caused relaxation in smooth muscles. Caffeine also acted as a competitive ‘antagonist’ by receptors of adenosine within the therapeutic concentration range. Some other inferior effects of caffeine included inhibiting “prostaglandin synthesis” and the reduction of the breakdown of catecholamine’s. Low concentrations of caffeine suppressed the outbreaks of adenosine in the brain. Adenosine decreased the rate of neuronal transmission and inhibited synaptic transmission as well as neurotransmitters. The turnover number of a batch of neurotransmitters also increased. Furthermore, caffeine also lead to the inhibition and blockage of adenosine receptors which lead to “potentiation of dopaminergic neurotransmission.” Various clinical tests have been carried out and revealed that caffeine stimulated the ‘autonomic nervous system and increased alertness.” Caffeine also showed the relief of sleepiness. One the other hand, trials revealed that caffeine had no effect on the ‘arousal of memory.” Further studies are required to understand the long and short term effects of caffeine as it applies to the nervous system and so the human system as a whole.

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I choose this topic because I love caffeinated drinks and I wanted to learn exactly how it affects my body. This article revealed some interesting facts about coffee. I am a frequent coffee consumer and I always thought it had an effect on memory. It showed me a wider understanding of caffeine with respect to the benefits but it did not reveal any disadvantages with consuming too much caffeinated drinks. Presume away but caffeinated drinks have kept me up in times of need and so i dedicated my first review to understanding how it really works. That said, the relevance of choosing the topic at hand was to portray my urge to understand how certain “necessities” work in the body. Thank you for reading and have a productive day.

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Topic 2 Reflection: “Bacteria can Morph Host cells into Stem cells”

Reference:

“Bacteria can Morph Host Cells into Stem Cells,” Medical News Today, accessed on March 24,2013, http://www.medicalnewstoday.com/articles/255150.php

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Upon studying what happened to mice infected with “leprosy bacterium,” scientists have discovered that at early stages prior to infection, the leprosy bacterium protected itself from the immune responses from the host by be cloistered away in Schwann cells. Upon infection, the leprosy bacterium then went ahead to reprogramme the host cell into stem cells, leaving the host (mice) unprotected. This then prevented (Schwann cell) the nervous transmissions to the brain. The new stem cells had the ability to now transform into any cell which helped the leprosy bacterium to go from the nervous system to other systems. Astonishingly, they also discovered that the leprosy bacteria was able to trick the immune system and thus helped the bacteria in spreading. This was done through the secretion of proteins called “chemokines” which gathered immunity cells to obtain the bacteria and disperse it to other parts of the body system. This was the first discover in terms of a bacteria transforming mature cells into stem cells. Researchers were interested in this new mechanism because this process did not have the potential risks of developing tumors which was always a result otherwise when scientists tried to develop stem cells in the lab. Scientist revealed that they would get stem cells through means of the leprosy bacterium and then get rid of the bacterium using antibodies so the new stem cells could be inserted to tissues that have underwent degeneration through diseases. Furthermore they planned to use this knowledge, in order to improve synthetic stem cell creation in the lab as well as applying the knowledge to other diseases.

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I choose to reflect upon this topic because I saw substantial relevance as it pertains to my field of interest. Many breakthroughs have been discovered and this is no exception. I have definitely learned something interesting however I would have liked to know how exactly the bacteria works as in converting the host cells to stem cells. I understand that it is a new discovery (article posted on 19 January 2013) and such more research is needed to understand how it works at the biochemical basis. The significance of this topic, well could be found in my first ever post. As I stated at the beginning, I would like to study microbiology in various aspects and for this, i see it fascinating and paramount to keep up with the “micro-trends” in the world. Some people prefer the “newest and latest” materials in the world today but i prefer to keep up with the new developing trends of the micro-world. Thank you for reading and have a blessed day.

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Identification of Lactose positive and Lactose negative Bacteria using MacConkey agar

mad_scientist

MacConkey agar contains bile salts and crystal violet to inhibit most gram + organisms.Lactose is a carbohydrate that may be used as a nutrient. The utilization of lactose is important in identifying gram negative rods. Lactose + means that the organism can use lactose as an energy source whilst lactose – means they cannot. Differential media is used to distinguish organisms from each other based on a reaction that occurs as they grow. Lactose + organisms ferment the lactose and produce acid whilst lactose – organisms do not produce any acid as they grow. MacConkey agar contains a pH indicator called neutral red that detects the acid production in lactose + organisms. As lactose + bacteria produce acid, it causes the pH to drop and where neutral red is absorbed and the colony turns red.

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Lactose – bacteria remains colorless and translucent as they do not produce any acid.

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Reference:

“Identification of Lactose Positive and Lactose Negative Bacteria using MacConkey Agar,” Wisc-online, accessed on April 10, 2013,  http://www.wisc-online.com/objects/ViewObject.aspx?ID=MBY701.

BioTork develops xylose-fermenting yeast for ethanol facilities

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After nearly two years of collaborative research, biotechnology company BioTork LLC and the National Corn-to-Ethanol Research Center have developed a yeast strain capable of fermenting the xylose found in ligno-cellulosic biomass in a commercial-scale environment.Xylose is the second most abundant sugar in ligno-cellulosic biomass but cannot be fermented by Saccharomyces cerevisiae, the predominant yeast used in ethanol production. The USDA had previously engineered a strain of S. cerevisiae capable of fermenting xylose, but the genetic engineering negatively impacted the strain’s growth rate, making it inapplicable for use in an industrial environment.  BioTork utilized proprietary continuous culture technology developed by Florida-based Evolugate LLC as part of an adaptive evolution method to essentially teach that strain to grow under industrialized settings. Xylose was placed in a medium with the yeast strain and the fittest yeast cells were then selected and re-introduced to increasing proportions of xylose over the course of several months until they eventually learned to grow on xylose alone.“While improvements to the growth rate and initial scale-up of its performance in an industrial setting are underway, this strain has the potential to be one of the first economically viable xylose-fermenting strains, and represent a fruitful combination of genetic engineering and adaptive evolution,” Tom Lyons, BioTork chief scientific officer said in a news release.Because D-xylose comprises up to 30 percent of cellulosic biomass, BioTork’s yeast strain could be applied to various types of biomass for cellulosic ethanol production but one of the first applications envisioned for the yeast strain, currently known only as SC48-EVG51, is to produce ethanol from distillers dried grains (DDGs) at existing corn ethanol facilities. According to BioTork, if the glucose and xylose in distillers grains were converted to ethanol, producers could increase their ethanol output by 10 percent without increasing their capital expenditures. “The sugar xylose represents close to 18 percent of the dry weight of distillers grains, and our partnership with NCERC has mostly solved the way to ferment it,” said Ziad Ghanimi, public relations manager at BioTork. While the U.S. ethanol industry is currently facing a domestic blendwall, Ghanimi noted that there continues to be demand internationally for increasing amounts of ethanol, which could provide the market for additional U.S. capacity.BioTork does not plan to make the newly developed yeast commercially available in one standard strain for distillers grains-to-ethanol production, simply because the chemical composition of distillers grains varies from plant to plant, Ghanimi said. Instead, the company will optimize the strain for use with each specific product. Licensing fees will likewise vary, he said.

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referenced;

“BioTork develops xylose-fermenting yeast for ethanol facilities,” Ethanol producer magazine,   accessed on April 10 2013,http://ethanolproducer.com/articles/8766/biotork-develops-xylose-fermenting-yeast-for-ethanol-facilities.

Managing Wild Yeast Contamination in Fermentation for Alcohol Production

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Introduction

Ethanol fermentation is a complex biological process for the production of alcohol from sugar.  Alcohol is widely used for many different purposes.  Yeast, under anaerobic conditions, convert glucose to ethanol.  The stoichiometric equation for the production of alcohol by fermentation is given below:

C6H12O6        ——————> C2H5OH   + 2CO     -265kcal /kg cal

Glucose                             ethyl alcohol     carbon dioxide

It is a non-sterile process.  The presence of undesirable microorganisms in the process can not be completely avoided.  The raw material used for fermentation itself shows the presence of contaminating microorganisms.Normally 60-70% of the production cost is contributed by the feedstock in alcohol production.  Owing to the increasing demand and cost of molasses, improving and maintaining the quality of the fermentation process has become a crucial factor.

 

What is Wild Yeast?

Yeasts, other than the elliptical culture yeast, are called as wild yeast.  Wild yeast occurs naturally in the raw material and produce small amounts of alcohol (Webster’s, 2004).  Report suggests that they can be harder to detect and control than bacteria (Fal, 1994). Generally the wild yeast Dekkera and Brettanomyces are observed inhabiting molasses source.  They are genetically identical but Dekkera is the sporulating form of this yeast (Goode, 2003). Dekkera species can also be fermentative yeast capable of producing alcohol. Brettanomyces grows and ferments slowly and can ferment in low levels of fermentable sugars (Kelly, 2003; Lansing, 2004).

Sources of Contamination

Wild yeast is considered to be indigenous and found in the air and on surfaces (Tessier, 2004).  Wild yeast primarily comes through molasses in the fermentation process because of the presence of fermentable sugars.   Solid addition as well as liquid streams entering the process can be a source of wild yeast contamination (Ingledew, 2001).

Problems Caused by Wild Yeast Contamination

A.   Scum Formation: What is Scum?

Wild yeast contamination is commonly observed in wine fermentation.  Little research has been performed on the topic of yeast contamination in molasses based fermentation.  Controversy surrounds the subject of scum formation as microbiologists and engineers debate whether the wild yeast itself is responsible or morphological changes in the cultured yeast under unfavorable conditions.In the fermentation process, yeast multiplies in the form of heavy branched structure.  This branching yeast comes at the top of the fermentor.  The sparging of air in the fermentor helps the branched yeast to rise to the top and form a thick layer called the ‘scum’.The scum formation in the fermentor traps gas bubbles inside the branched structure thereby building pressure in the fermentation vessel. Excessive and uncontrolled scum in the fermentation process is the major problem caused by wild yeast.  This scum cannot be reduced by the addition of anti-foaming agents.  The process needs to be stopped after a few weeks due to a loss of efficiency and excessive overflow of mash from the fermentors.  There is washout of culture yeast and a gradual drop in the alcohol concentration in the fermentation process. According to Lorenz et al. (2000), the morphological changes in the culture yeast (Saccharomyces cerevisiae) leads to filament formation under unfavorable fermentation conditions.  This causes scum formation and foaming.  The budding yeast S. cerevisiae, starved for nitrogen,  differentiates into a filamentous growth form.   In nitrogen poor conditions leucine, the precursor of iso-amyl alcohol (a chief constituent of fusel oil) can also induce elongation of cells.  Ceccato-Antonini and Paula Christina (2002) suggested genetically controlled morphological changes and filamentous growth in response to Iso-amyl alcohol.

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B. Reduced Efficiency

According to Lorenz et al, (2000), the reduction in efficiency is attributed to the presence of wild yeast and bacterial contamination in the input to the fermentor.  Due to heavy foaming, juice losses result in the loss of alcohol.   The efficiency reduction depends upon the extent of contamination.

Isolation and Identification of Wild Yeast

There are various methods to identify and differentiate wild yeast, two of them are:

  1. Heat resistance method: Suspend the yeast in sterile water and heat to 53 °C for 10 minutes. Then take a viability test to measure the survival rate.Normal cultured yeast will not survive this test. The wild yeast isolated is heat resistant.
  2. Microscopic examination: Since detection and isolation of wild yeast is a problem, the common method is plating followed by microscopic evaluation.  The presence of branched structure under the microscope reveals the presence of wild yeast (Lansing, 2004).

Differentiation and identification of various yeast strains along with contaminated wild yeasts has been done using selective culturing methods based on selective chemicals, fermentation tests, and other methods.  A synthetic medium, such as lysine, is used for the isolation and enumeration of wild yeasts (Oxoid, 2004).  Normal S. cerevisiae or carlsbergensis strains cannot utilize lysine whereas many other wild types of yeast do (Morris, 1957).  Differential media such as SDM (Schwartz Differential Medium) and WL (Wallerstein Laboratory nutrient agar, Green, 1950), Lysine medium (LYS), Lin Wild Yeast Medium (LWYM) and UBA (Universal Beer Agar) can be used to isolate yeast strains from different sources (Ceccato-antonini et al., 2004; brewingtechniques, 2004). Crystal violet in the medium will inhibit the growth of cultured yeast and allow the growth of Saccharomyces spp. which will not be detectable on lysine medium (Fal, 1994).  The uses of selective chemicals like actidione (cycloheximide), lysine, or cupric sulfate (LCSM) are commonly used for isolating wild yeast. Recently modern techniques like DNA fingerprinting and RT-PCR analysis have been applied to successfully detect and enumerate lower levels of wild yeast in a much shorter time (Morris, 1994; Cocolin et al., 2004; Lansing, 2004).

Wild Yeast Management

A. Killer Yeast Strains

There are widespread occurrences of killer phenotypes in yeasts in alcoholic fermentation.  Many of these fermentative processes use non-pasteurized medium, which can enhance the predominance of wild yeast.  These contaminants can contribute to the fermentation rate decrease or blockage, increases in acidity, fusel oil production, and an overall decrease in ethanol production.   Isolation of killer yeast strains from the ethanol process is imperative for good yields.  The presence of cultured yeast with “killer” tendencies against wild yeast will help overcome the problem of excess growth of wild yeast contamination during the process (Ceccato-Antonini et al., 2004). Sugar cane molasses are normally pasteurized or decontaminated in order to reduce the amount of microorganisms appearing during its production, transportation, or storage.  Usually, heating the molasses above 76.6°C reduces lactic bacteria but also decreases viscosity and precipitates calcium.  High concentrations of suspended solids also makes pasteurization inefficient.   Furthermore, the availability of good quality molasses depends on the efficiencies of the specific sugar refineries, weather conditions, and harvesting techniques.   Unfortunately, these variables lead to an inconsistent supply of good quality sugar cane molasses and a constant dilemma for sugar cane molasses distillers (IFT, 2003). It is not altogether necessary to sterilize molasses.  However, there are advantages in terms of alcohol yield and purity when pretreating the molasses.  Treatment of the molasses solution by means of heat and sulfuric acid will precipitate undesirable salts and help to improve the purity of alcohol obtained and reduce the amount of scaling.

B. Sulfur Dioxide and Sulfite Solutions

The principle source of SO2 is sodium bisulfite or potassium metabisulfite.  Sulfite is available in powdered form but because of its elevated level of toxicity, its use is limited.  Potassium metabisulfite contains approximately 55% SOby weight.  This free SO2 kills the microorganisms (Kelly, 2003). Culture yeast is generally tolerant to SO2, but at higher concentrations it shows loss of viability.  SO2 is toxic and requires appropriate handling. SO2 binds to various compounds present in molasses or juice and pulp or the compound formed during fermentation.  Hence the dose should be optimized so that the deleterious effect on wild yeast is achieved without harming the culture yeast.  The concentration should be sufficient to overcome the efficiency loss caused by binding to the compounds during fermentation.  Generally the dose should be optimized in order to achieve the proper results.  The total SO2 added to any wine is under 100 PPM. Sodium bisulfite and potassium metabisulfite are corrosive and will degrade upon exposure to oxygen and moisture.  Purchasing should be done depending upon the climatic conditions.  The storage should be done in airtight containers preferably glass containers.  Precaution should be taken while handling sulfites as it destroys mucus membranes including lung tissues (Nashwoodwinery, 2004).

C. Process Parameters

Control of Infection

As discussed earlier, pasteurization of molasses can help control wild yeast and bacteria.   Contaminants generally occupy surfaces covered with deposits of starch, sugar, protein or mineral rich materials.  Contaminants thrive in places that are hard to clean such as, porous surfaces, cracks, sharp angles, corners, gaskets, valves, pressure gauges, in-place thermometers, and pump packing.  Deposits in these areas protect the organisms in them from heat and sterilizing solutions.

Adequate Yeast Cell Population

According to Abbott et al., (2004) the growth rate of Dekkera wild yeast is lower than culture yeast S. cerevisiae and hence their ability to compete with S. cerevisiae is hindered in batch fermentation.  Due to different reaction rates Dekkera will produce different end product as they ferment the juice sugars to alcohol (Aris, 2002). Considering this fact, the cell concentration of the culture yeast can be higher but suppressed if wild yeast outnumbers the culture yeast.   Recycle of yeast builds up higher population of yeast in the fermenting mash, thus giving higher productivity as well as robustness of operation (Deshpande, 2002).   There are processes that would allow the yeast cell concentration in the fermentation process to be increased.  Yeast recycling is one of the most common methods.  This process maintains desirable cell concentration for healthy fermentation.  However, maintaining the population of these contaminants at lower levels and the use of proper operating procedures is key to the fermentation process.

Temperature

Online cooling and mixing of molasses is important to avoid the caramelization of sugars.  Molasses, if not cooled properly, helps the growth of heat stable microbes that affects fermentation.  At higher temperatures, generally in the summer, wild yeast is produced in high quantities. The processing of fresh molasses usually results in a high microbial count in distillery fermentation.   Sulfurous gases can inhibit yeast.  Similarly fresh molasses has a high foaming tendency as well as a high buffering capacity.  It also contains high levels of suspended sludge.  This is why fresh molasses is stored for at least a month before use in a distillery.  Cooling and frequent mixing by recirculation during storage helps to avoid internal combustion and caramelization that can deteriorate quality.  Excessive storage time (more than six month) on the other hand can reduce the fermentable sugar content slowly.  Molasses is stored in steel tank to prevent contamination.  In order to control the effects of aging, recording the production span, life, and operating with “first in first out” principle are normally standard procedure. The sugar factory process along with handling, storage and transportation of molasses is vital to managing the contamination of wild yeast. As discussed earlier, the presence of sugar in the medium favors growth of wild yeast along with nutrients.  Contact with water and pockets of dilution in bulk stock can give rise to high microbial flora.  Contact between soil with molasses also should be avoided.  It is necessary to handle and store via a protective environment (Bhutto, 2004; Lansing, 2004).

Nutrient Supplement

The vital risk factors for Dekkera growth include residual sugar and nitrogen present in the fermentation medium (Goode, 2003).  Nitrogen is supplemented in the form of Diammonium Phosphate (DAP) or urea to control fermentation.  This can lead to the excess growth of Dekkera due to the presence of excess nitrogen left over during fermentation.  Hence the quantity of nitrogen added to the fermentation process should be adequate only to help growth of culture yeast. According to Lorenz (2000) S. cerevisiae can change its morphology due to an inadequate nutrient supply.  In this case, the proper supply of nitrogen should be used to overcome the morphological changes in culture yeast.

Air Filtration

Airborne dust can vector microorganisms (Fal, 1994).  The most efficient means of reducing wild yeast coming from the air is to use a filtration membrane of 0.45 microns.

Conclusions

Wild yeast contamination can lead to serious problems including scum, foaming, and the loss of yield in fermentation.  The widespread occurrence of these problems challenges the industries and scientific community to look forward and contributes to the management of wild yeast contamination.  Further study of this topic is obviously warranted.

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Reference:

“Managing Wild Yeast Contamination in Fermentation for Alcohol Production,” Che Plus, accessed on april 10    2013,http://www.cheresources.com/wild_yeast_contamination.shtml

Leave me alone you poison

Electron transport inhibitors

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ETS inhibitors act by binding somewhere on the electron transport chain, literally preventing electrons from being passed from one carrier to the next. They all act specifically, that is, each inhibitor binds a particular carrier or complex in the ETS. Irreversible inhibition results in a complete stoppage of respiration via the blocked pathway. Competitive inhibition allows some oxygen consumption since a “trickle” of electrons can still pass through the blocked site. Although it allows some oxygen consumption, competitive inhibition prevents maintenance of a chemiosmotic gradient, thus the addition of ADP can have no effect on respiration.Whatever the mechanism of inhibition, an electron transport inhibitor can block respiration specifically along the NADH pathway, along the succinate pathway, or along the pathway that is common to both routes of electron entry. Careful addition of inhibitors to mitochondria on specific substrates can reveal the sites of inhibition. Some combinations of inhibitors enable demonstration of alternative entry points to the electron transport system.

Rotenone

Rotenone is still used as an insecticide, but is not available for general use. It is toxic to wildlife and to humans as well as to insects. The location of inhibition by this competitive inhibitor of electron transport can be worked out by testing its ability to block respiration via the NADH versus succinate pathway.

Antimycin

The antimycin that we use in research was formerly known as antimycin A. The latter term has been dropped since only one antimycin is used in the literature. The binding site for antimycin can be narrowed considerably using combinations of substrates inlcuding succinate, NADH or glutamate, and the dye TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine) along with ascorbic acid.

Cyanide

Cyanide is an extremely effective reversible inhibitor of cytochrome oxidase. A concentration of 1 mM KCN is sufficient to inhibit oxygen consumption by mitochondria from a vertebrate source by >98%. For a nominally 2 ml chamber, a convenient concentration for the stock solution would be 0.5M (20 µl produces a 2.5 mM final concentration). Mitochondria from some sources have cyanide resistant pathways. KCN solutions are volatile, so that a dilute solution left open to the atmosphere will quickly lose its potency. Concentrations greater than 1 mM have been known to cause uncoupling. In the presence of TMPD we have seen a dramatic increase in oxygen consumption upon the addition of excess cyanide, using a Clark electrode. Indications were that a non-biological mechanism was responsible. Cyanide is one of the most deadly compounds in a laboratory. Stocks of the dry chemical should be stored under lock and key. As we know from the Tylenol incidents of a number of years ago, a 500 mg capsule can hold enough cyanide to kill a person. Because of its volatility, exposure to fumes from large quantities is hazardous.

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Malonate

Malonate (malonic acid) has long been known to inhibit cellular respiration. Among the key observations made in the 1930s investigations into the nature of cellular respiration was that the addition of fumarate, malate, or oxaloacetate to cell preparations resulted in the accumulation of succinate in the presence of malonate. Malonate is in fact a competitive inhibitor, and although we treat it as an inhibitor of electron transport it really is an enzyme inhibitor.

Uncoupling agents

Uncoupling is defined as a condition in which the rate of electron transport can no longer be regulated by an intact chemiosmotic gradient. The condition is differentiated from electron transport inhibition by the fact that in the latter case, bypassing the block can restore the gradient. In uncoupling, the electron transport system is uninhibited due to complete and irreversible dissipation of the chemiosmotic gradient.

2,4-Dinitrophenol

The compound 2,4-dinitrophenol (DNP) acts as a proton ionophore, that is, it binds protons on one side of a membrane, and being fat-soluble it drifts to the opposite side where it loses the protons. Actually, the associations/dissociations are random, but the probability of binding is greatest on the side of the membrane with greatest proton concentration, and least on the side with the lesser concentration. Thus, it is impossible to maintain a proton gradient with sufficient DNP in the system.DNP is known to have mixed actions, that is, it produces other effects in addition to uncoupling. DNP gradually inhibits electron transport itself as it is incorporated into mitochondrial membranes. The effects appear to depend on concentration of DNP and of mitochondria, and vary from one preparation to the next.Back in the 1930s DNP was touted as an effective diet pill. Indeed, the uncoupling of electron transport from ATP synthesis allows rapid oxidation of Krebs substrates, promoting the mobilization of carbohydrates and fats, since regulatory pathways are programmed to maintain concentrations of those substrates at set levels. Since the energy is lost as heat, biosynthesis is not promoted, and weight loss is dramatic. However, to quote Efraim Racker,”the treatment eliminated not only the fat but also the patients,…This discouraged physicians for awhile…” It is not a good idea to mess with cellular metabolism.

Carbonyl cyanide p-[rifluoromethoxyl]-phenyl-hydrozone (FCCP)

This agent is, in fact, a pure uncoupler. It acts as an ionophore, completely dissipating the chemiosmotic gradient, leaving the electron transport system uninhibited. It is also expensive.

Oligomycin

Oligomycin, an antibiotic, acts by binding ATP synthase in such a way as to block the proton channel. That is the mechanism by which oligomycin inhibits oxidative phosphorylation. Experimentally, oligomycin has no effect on state IV respiration, that is, it has no direct effect on electron transport or the chemiosmotic gradient. On the other hand oligomycin prevents state III respiration completely. To draw the conclusion that an agent is an inhibitor of ATP synthase (inhibitor of oxidative phosphorylation), the above conditions must be demonstrated experimentally and unequivocally.It takes awhile for the effects of oligmycin to show up. Attempts to interrupt state
III respiration by adding oligomycin may fail because of the delay.

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reference; “Electron Transport Inhibitors,” Experimental BioSciences, accessed on April 10 2013, http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitopoisons.html.

 

The Citric Acid Cycle Is Controlled at Several Points

The rate of the citric acid cycle is precisely adjusted to meet an animal cell’s needs for ATP.The primary control points are the allosteric enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase

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Control of the Citric Acid Cycle. The citric acid cycle is regulated primarily by the concentration of ATP and NADH. The key control points are the enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.

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Isocitrate dehydrogenase is allosterically stimulated by ADP, which enhances the enzyme’s affinity for substrates. The binding of isocitrate, NAD+, Mg2+, and ADP is mutually cooperative. In contrast, NADH inhibits iso-citrate dehydrogenase by directly displacing NAD+. ATP, too, is inhibitory. It is important to note that several steps in the cycle require NAD+ or FAD, which are abundant only when the energy charge is low.

A second control site in the citric acid cycle is α-ketoglutarate dehydrogenase. Some aspects of this enzyme’s control are like those of the pyruvate dehydrogenase complex, as might be expected from the homology of the two enzymes. α-Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, α-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced when the cell has a high level of ATP.

In many bacteria, the funneling of two-carbon fragments into the cycle also is controlled. The synthesis of citrate from oxaloacetate and acetyl CoA carbon units is an important control point in these organisms. ATP is an allosteric inhibitor of citrate synthase. The effect of ATP is to increase the value of KM for acetyl CoA. Thus, as the level of ATP increases, less of this enzyme is saturated with acetyl CoA and so less citrate is formed.

reference: “The Citric Acid cycle is Controlled at Several Points,” NCBI, assessed on April 10,  2013, http://www.ncbi.nlm.nih.gov/books/NBK22347/.

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