effect and Pasteur Effect will be here presented following its foundations. ... 1- Pasteur Effect â Warburg Effect - Key fast metabolic regulation of glycolysis during.
Warburg Effect or Pasteur Effect revisited with biochemical and biological links to cancer. José Eduardo de Salles Roselino Bioquímica – FMRP - USP Ribeirão Preto SP Bow, stern, port, starboard definitions for a boat, were written on a “magic small piece of paper” consulted by an old captain, until he was about to retire. He was using the information of this small paper always immediately before imparting successful orders to his crew about how to face huge storms with his ship during his professional life. A lot of molecular biology data seems to have being taken without careful definitions of some very important references or general aspects of biology. Among these general aspects, homeostasis seems to be one disregarded or misinterpreted by loose definition. Warburg effect and Pasteur Effect will be here presented following its foundations. Models microorganisms will be considered according to properties shared with complex animal cells and cancer (a word that includes a large number of very different conditions) will be mentioned regarded lost cell differentiation and ATP production using anaerobic glycolysis even under aerobiosis. 1- Pasteur Effect – Warburg Effect - Key fast metabolic regulation of glycolysis during Aerobic-Anaerobic transition. Intracellular versus homeostatic regulation. “Regulation of internal milieu” was one key aspect of the role of Claude Bernard in the continuation of Pasteur´s work (Biological chemistry studied outside the cells - in cellular external microenvironment). He has made very important discovery concerning the role of liver in regulation of blood glucose levels among other aspects. His work was latter rediscovered by Walter Cannon that coined the expression homeostatic regulation for everything that is under a very complex form of regulation outside the cells. Homeostasis as a special type of regulation is clearly absent in microorganisms (1 - 3). Recently, taking into account that great biochemical discoveries were made with the use of techniques from biochemistry and genetics research made in microorganisms’. For instance, chemiosmotic hypothesis of ATP production in mitochondria (P. Mitchell), or the role of rhodopsin in harvesting chemical energy from light (photons), it was assumed that every question about biology must also have its answer found in microorganism’s biochemistry as well. Following a line of consequence, homeostasis, something absent in microorganism regulation (3), lost its rigorous definition and was “adjusted” to become adequate for the new biochemical reasoning that ignores differences between complex animals and microorganisms. Regulation of the “internal milieu” (extracellular) was transformed into regulation of the “intracellular” even for those simpler forms of microorganisms that do not have real cellular structures. In addition, mitochondria an intracellular organelle, could become directly involved in homeostatic regulation of some of its components. When the original and correct meaning is restored, some aspects of the logic of biology for fast regulatory responses become amenable to our understanding in a form that indicates great
differences in comparison with slow regulatory responses involved in adaptation and/or complex developmental phenomena. For instance, we must take into account that only glucose can provide intracellular maintenance of ATP levels, through a key intracellular regulatory mechanism, in absence of oxygen or in the absence of the capability to use oxygen as an electron acceptor. In absence of oxygen and/or absence of mitochondrial function, only the anaerobic glycolysis pathway can provide a continuous form of harvesting chemical energy available in glucose molecules in order to produce ATP. When a facultative organism is changed from aerobic environmental condition to anaerobic condition the organism is rapidly informed about the environmental change and starts immediately and at the same time, two different types of regulatory response to the environmental change. One is a fast response triggered by changes in its functional regulatory proteins the other is a change in its proteins that regulate gene expression pattern. In case, the environmental change is rapidly reverted only the first kind of regulatory response explain the biological changes observed that occurs and preserves the organism life. These fast changes that happens with very limited use of energy for the response - Mainly with the use of regulatory conformational change of proteins, that does not require ATP use. The definition of anaerobic glycolysis must thus, be linked with the form of harvesting chemical energy to produce ATP and not with the absence or presence of oxygen. For instance, as will be mentioned, red blood cells use anaerobic glycolysis to produce ATP in presence of oxygen transported by the same red blood cells. We should not call the conversion of carbon from glucose into carbon of lactate molecules as aerobic glycolysis just because it is happening in the presence of oxygen. It is anaerobic glycolysis happening in the presence of oxygen. Cancer cells, uses anaerobic glycolysis despite the presence of oxygen and even with the capability to perform aerobic glycolysis as well. This form of ATP production, using anaerobic glycolysis is what happens in normal mature red blood cells, that lacks mitochondria and nucleus and yet, during 90-100 days will perform their vital biological function but under a stringent dependency of glucose levels in the blood (whose maintenance results from homeostatic regulation). Despite its key role in oxygen transportation, red blood cells are performing anaerobic glycolysis or the metabolism of glucose to lactate regenerating NAD from NADH + H, at the last step of this metabolic pathway during the pyruvate-lactate conversion. This last step replaces the requirement for the respiratory chain function in mitochondria that recovers NAD from NADH + H under aerobic metabolism. Even in case we did not acknowledge that neurons also have an almost absolute requirements for glucose, red blood cells dependency would indicate a key regulatory target for homeostatic regulation of blood glucose levels that cannot be studied in microorganism´s biochemistry. Similarly, this general reasoning can be applied to tumor cells. Some stem cells growth in presence of almost normal oxygen levels and have functional mitochondria however, they will develop into cancer cells and present as a biochemical mark its active anaerobic glycolysis producing lactate from glucose instead of carbonic gas and water as aerobic glycolysis does. In the red blood cells, lack of mitochondria explains “anaerobic glycolysis”; in tumor cells, the drift away from normal metabolic regulation is a matter of dispute. Is it derived from metabolic condition that favours genomic changes or it is a metabolic disorder caused by genetic changes?
In case the dogmatic reasoning, that considers that in biology all the “information” and not only the genetic information follows that arrow that goes from DNA through RNA to proteins, the second option is the answer. However, environmental and microenvironmental factors must be taken into account as “anti-genetic factors” (environmental factors) and not as merely “epigenetic” ones when genetic roots of the problem are just a little bit more than a speculation. It is clear that red blood cells and neurons require maintenance of blood glucose. Therefore, the development of our nervous system must have being dependent upon the very complex regulatory system (neuronal plus endocrine, paracrine and autocrine regulatory mechanisms) placed above intracellular metabolic regulation. Alternatively, in very general terms, the development of our neural system is dependent upon various forms of differentiated cell function that preserves the level of blood glucose in what is considered its normal range of variation after meals and during fasting. One aspect of homeostatic regulation as described by Walter Cannon (2) was that it might present an oscillatory pattern inside a normal range of variation of the regulated parameter. Since, as it will be shown latter, it cannot work in instantaneous mode everywhere in the body; homeostatic control requires longer times than fast intracellular metabolic regulation even when fast regulatory mechanisms (the ones that do not require changes in gene expression) are used by very different set of cells at the same time. Furthermore, it is not possible to imagine that evolution of any protein involved in maintaining this mechanisms could follow a one gene plus another gene mutation mechanism as is usually, also wrongly, presented for the development of biochemical pathways in microorganisms. (For those interested in critical view of clonal evolution in microorganism’s metabolic pathway development, it is worth indicate that evolution of some enzymes cannot be understood without taking into account joint evolution of other enzymes both for catalytic as well as regulatory mechanisms – Unregulated life equals death in evolution terms; It will not be here discussed). Homeostatic regulation requires several levels of complex integration that are far beyond this form of reasoning and that here and now, will not be addressed in detail. In short, maintenance of blood glucose levels for example, will depend upon one kind of proteins of a cell type that uses blood glucose. In addition to that, will depend upon a completely different set of proteins of another kind of cell for example, which can release glucose on the blood either from liver glycogen stores or from the cells of the intestine or from the liver after gluconeogenetic activity. Therefore, even during a period of time when these mechanisms were using fast regulatory response its results in homeostatic regulation cannot be perceived immediately in a reductionist form that would take into account only a single protein. However, in a very simple initial form, it is also possible to consider that every cell independently of its differentiated function, have very fast regulatory mechanisms necessary to maintain its intracellular ATP levels without depending on changes of gene expression (4). In the case of hearth muscle cells that do not have any resting period, this is indisputable. Heart cells are always using any metabolite in order to produce Acetyl-coA for mitochondrial metabolism. Including, blood lactate in reversed last step of anaerobic glycolysis may be converted into pyruvate in order to burn it inside its heart cell, Krebs cycle mitochondria. Mostly, the regulation of carbon flow will use simple displacement of chemical equilibrium in
order to continuously produce Acetyl-coA that will be joined to oxaloacetate producing citrate in Krebs cycle. Any critical role for glucose metabolism in the case above presented, is the anaplerotic maintenance of oxaloacetate by pyruvate carboxylase in case oxaloacetate is not sufficiently recycled in the usual Krebs cycle carbon flow or another indirect form of anaplerosis of oxaloacetate levels is taken into account. Therefore, minimal amounts of carbon originated from the glucose molecule could be indirectly required to maintain ATP aerobic production. In this scenario, the two major initial control points of glycolytic flow in this case, are the two first bottleneck enzymes hexokinase and phosphofructokinase. The first enzyme will be controlled by its product glucose 6 phosphate levels. The second enzyme, phosphofructo kinase, will be controlled in very sophisticated way. For example, levels of citrate, indicating as a form of intracellular information that the Krebs cycle is fully operational, will change the enzyme conformation that will then, be inhibited by normal levels of ATP that are not inhibitory in absence of citrate. With various levels of efficiency, this inhibition is found in phosphofructokinases of other tissues or cells. For instance, even in mature red blood cells that do not have mitochondria and Krebs cycle to internally produce citrate but, may be stored in presence of citrate used as anticoagulant in blood banks, this may cause through inhibition by normal ATP levels a slow (low temperature) but continue use of 2,3 phosphoglycerate. The reduction in the level of this key red blood cell metabolite will cause hemoglobin malfunction due to an abnormally high affinity for oxygen that will not be released at peripheral tissues by the hemoglobin persistent R conformational state. How can be understood this very sophisticated form of regulation of phosphofructokinase regarding intracellular control of ATP production? It has no meaning or at least a very limited one; except for the flexibility of the heart metabolism as source of aerobic ATP is concern. However, the same pattern of inhibitory sensitivity to ATP controlled by citrate levels, acquires biological meaning when the homeostatic control of blood glucose levels therefore, a control over a chemical kept outside the cell is considered. In this case, favouring as source of AcetylcoA, everything instead of pyruvate formed from glucose, this control is a form of sparing glucose for those cells that does not have any alternative sources of ATP, as is the case of Red Blood Cells (RBC) or the case of neurons. This is a clear example that indicates a necessary antireductionist form of biochemical reasoning despite of the fact that biochemistry is a reductionist scientific endeavour. In addition, returning to the key aspects of Warburg Effect – Pasteur Effect, in case ATP an intracellular production is concern, the transition from aerobic metabolism to anaerobic condition, the sole enzyme that can replace mitochondria as ATP producer is a key regulatory enzyme of glycolysis that is not a bottleneck - Pyruvate Kinase. Pyruvate kinase is a very complex regulatory enzyme of glycolytic flow in some systems as for instance the liver that will be considered latter. However, as an unusual mark, it is always presented in large amounts (contrary to what is found as a rule followed by any other regulatory, bottleneck enzyme). Most of the enzyme is kept in T (tense) conformation that is less active than the R conformation. However, it changes immediately its conformation to R (active form) immediately after increase in phosphofructokinase activity that increases Fructose 1,6 diphosphate levels and Phosphoenolpyruvate levels. Both agents, cause fast change from T (tense inactive) to R (relax active) conformation (R and T conformation as proposed by allosteric model of enzymes in Monod, Wyman and Changeux 1965 J. Mol. Biol., 12, 96 ) despite of the dogma that says that sequence determines protein conformation.
Therefore, when there is no mitochondrial ATP production by lack of oxygen or lack of mitochondrial function in presence of oxygen (virus or toxic agents) it is this enzyme, pyruvate kinase that replaces mitochondria as ATP producer with a very fast carbon flow since it will use a low ATP yield metabolic pathway (anaerobic glycolysis). This low ATP yield explains requirement for a very large metabolic flow together with the fact that it will receive double three carbon molecules from the six carbons of glucose, as two trioses. This detail about the required carbon flow allow us to understand why this enzyme is a regulatory anomaly in terms of not being a “bottleneck” regulatory step. It must have a fast mechanism that allows for a fast maintenance of ATP levels without any change in gene expression - Only changes in protein conformation. This general pattern of regulation is preserved in all cells types. Every decrease in mitochondrial ATP production is followed by increased carbon flow from glucose to lactate by a massive activation of pyruvate kinase. The key factor in the enzyme activation is the proportion of AMP, ADP and ATP required for maintaining the energy charge of all cells (4). The proportion of ATP, ADP and AMP that is observed throughout all kind of living cell makes changes in AMP levels a very sensitive regulator of anaerobic ATP production. For this key role, any small reduction in ATP levels is halved by Adenylate kinase using two ADP molecules to restore one ATP while producing one AMP. Therefore, every small decrease in ATP represents in percentage a great increase in AMP levels that are kept in very small amounts inside the cells by the same universal system that preserves energy charge in any kind of cell (4). Complementarily, ADP is an indicator for aerobic ATP production through RCR respiratory control (5). AMP minimal increase is the most powerful internal signal for increase in anaerobic glycolysis. This regulatory very fast change represents one side of the “Pasteur Effect” when activation of anaerobic glycolytic flow is addressed. The opposite side was already mentioned when the transition from high carbon flow observed in anaerobiosis moves to low carbon flow from glucose. This is observed after starting mitochondrial ATP production. The major reason is that the metabolism moves to a high ATP yield mitochondrial metabolism in aerobic condition. Mitochondrial metabolism is kept under Briton Chance´s RCR regulatory index (5). When plastic use of carbon is not needed, ATP is produced at expense of Krebs cycle and respiratory chain only to maintain cellular ATP levels. For the heart, a revision about cardioplegia shows in very detailed manner a series of events in seconds, minutes and hours that are started by interruption of the differentiated heart cell function that places heart cell in a drastic ischemic condition (6). Usually, this condition is prompted by medical decision after the injection of a high potassium chloride blood solution in the heart blood supply. This calls attention again to this key aspect, heart function depends upon intracellular ATP levels and upon extracellular K homeostasis (blood potassium levels). This last parameter depends upon digestive absorption and kidney excretion, that is also affected by hydration, acidic excretion (involving respiratory, kidney function) etc. Above all, the cardiac arrest caused by decreased or increased potassium levels under or above normal blood limits shows a way in which differentiated cell function (heart beating activity) can be stopped without killing the cell or destroying its genome. Another blow to the dogmatic reasoning heads. In another form: In the case of the change in blood potassium levels prompted by a medical manoeuvre, this reasoning is clearly supported by the fact that the medical doctor, aims at a better heart recovery after surgery. Not to kill his patient. In short, through preservation of ATP levels that otherwise would be spent in the muscle contractions he hopes the heart resumes differentiated function afterwards as soon as K levels return to normal levels. This is obviously a case in which, an information goes from outside the cell (high or low K-Potassium
blood levels) to the membrane and can stop differentiated cell function (heart beating) without killing the cell in order to produce this heart arrest. Therefore, it is a rather simple and straightforward fact that stands against the dogmatic reasoning that proposes – “All information in biology goes from DNA through RNA to proteins”. Other experimental observations also show that auto assembly, the most remarkable aspect of biology, is also affected by aerobic-anaerobic condition or by the pattern of metabolism. The information flow from DNA alone only renders a clear molecular picture in the case of undisputed inherited errors. It is a very serious error to consider that we can extrapolate the clarity of these circumstances (deterministic aspect of the inborn errors) to the entire biochemistry of medical conditions. Mathematical sophistication will not jump over the barrier of different regulatory functional domains in biology. Normal values in humans depends upon the completely functional range of several different sets of proteins that frequently work in opposite form regarding the resulting normal value range for every vital parameter. For instance, consider the blood potassium levels or blood glucose levels. Any reductionist view directed to a single protein or to a single set of proteins, for instance those involved in reduction of the blood levels will led to a partial result in case, those proteins that function in increasing the values of the same blood levels are not also considered. For a metabolic measurement made inside a single cell this problem also exists but the two sets of opposing proteins in normal condition are held in self-assembled organization therefore, receiving regulatory signals from the environment in concerted and simultaneous form. This is often seen in the biochemistry of microorganisms and is absent from the mechanisms of our homeostatic regulation. 2- Pasteur Effect – Warburg Effect in microorganisms However, as long as the differences in the general aspects are taken into account microorganisms have provided important models for the understanding of simpler aspects of the biochemistry of more complex systems. For an important example about the problem here considered – Pasteur Effect, we may use the illustrated words of Salomon Bartnicki-Garcia in 1963 in italics below (7). The text presented below, clearly indicates that the root of the reasoning about changes observed in living forms and metabolism when the effect of the presence or absence of air (oxygen) is compared are born out of the work of L. Pasteur. The link between aerobiosis and anaerobiosis transitions with the “Pasteur Effect” is what has led Warburg to coin the expression “Pasteur Effect” when the same reasoning was applied to the comparison of normal with cancerous cells submitted to the same aerobic versus anaerobic transitions. All living organisms are regions in the space that harvest matter and free energy changes from the environment and transforms it into order by a dissipative mechanism (moving heath to the environment). Therefore, living organisms depends upon the possession of a signal system that have among other properties an informational role required to convey a chemical description about the environmental condition where they are thriving (In the case here considered, aerobic or anaerobic environment). M. rouxii requires high levels of glucose in its growth media under anaerobiosis. Anaerobic glycolysis is the sole form of harvesting energy from the environment for this facultative aerobic microorganism when no oxygen is present in the environment and this metabolic pathway has low ATP yield in comparison with the one observed in aerobic condition. In aerobic metabolism, glucose carbons even indirectly flows
from glucose to carbon dioxide instead of to ethanol carbons in yeasts or lactate in human cells. On the other hand, this growth condition (anaerobic) does not shows any strong dependence upon mitochondrial metabolism. M. rouxii mycelium may growth in absence of glucose, using amino acids as carbon source as long as aerobic condition exists. However, it will depends upon the presence of mitochondrial function and will require an electron acceptor (usually is oxygen).
Figure 1
“In the development of many microscopic fungi, there are simple but well-defined examples of morphological differentiation that are amenable subjects for studying biochemistry of morphogenesis. In some of these cases, besides the convenience of rapid and facile cultivation in the laboratory, there is the important attribute of an exogenously controlled morphogenesis. Since cells are in direct contact with the environment, a close and reproducible control of form development is possible through proper adjustment of specific chemical or physical factors of the external milieu. Furthermore, if a well-defined and specific chemical agent is found to affect morphogenesis, it immediately constitutes a reliable tool to probe the biochemical machinery of the cell, in a guided search for processes underlying form development.
Mold-yeast dimorphism of Mucor has historic interest for the microbiologist. The phenomenon was used last century to dispel ideas on species transmutation and to illustrate, in a most convincing manner, the ready capability of microorganisms to adapt to different environments by modifying their cellular constitution. The morphological variability of Mucor was noticed over a century ago. At that relatively early stage in microbiology, the presence of drastically different morphologies in cultures of fungi led to erroneous interpretations. Thus, in 1857, Bail described what appears to be the first clear example of mold-yeast dimorphism in fungi. He observed that saccharine cultures of Mucor (Al. racemosus?) contained numerous spherical cells which multiplied by sprouting more spherical elements; he then concluded that the budding cells were ordinary brewers' yeasts, and that “Hormiscium cerevisiae” (former name for Saccharomyces cerevisiae) and Mucor were developmental stages of the same fungus. In 1861, Pasteur tacitly rejected such possibility of species transmutation, and some years later, on confirming Bail's observations, he provided a different interpretation. Accordingly, the morphology of the mold Mucor became yeast like as a result of oxygen deprivation. On the surface of liquid cultures, where air was plentiful, the fungus developed ordinary mycelial turfs, whereas in the depth of the same cultures, where aeration was insufficient, the fungus assumed budding yeast like shapes and conducted a strong alcoholic fermentation. Pasteur saw in Mucor a clear example of the capacity of an organism to adapt itself to a modified environment by modifying its own living machinery, an example which was more dramatic than that of S. cerevisiae, since in Mucor adaptation to "la vie sans l'air" involved conspicuous morphological as well as metabolic modifications.”
In the M. rouxii mycelium, it is possible to observe a mass effect similar to what is described as “contact inhibition” of growth found in Petri dishes monolayer grown cells. Malignant transformed cells does not shows contact inhibition. Contact inhibition represents a form of growth control derived from biological information that is generated outside the cell and moves toward the cell. Therefore, as also can be perceived in the case of potassium-induced cardiac arrest (cardioplegia), there is a flow of information that moves against the usual flow of genetic information. In order to clarify the difference between what is proposed by molecular biology dogmatic view, it helps to place proteins at the center stage instead of DNA regarding biological function. This renewed picture shows on the left, DNA as the source of genetic information and in the right side of proteins, the environment through the membrane as the source of the information that affects protein conformation, activity and function. Contact inhibition information flow goes from environment to proteins, clearly without causing any effect upon DNA base sequences - what would be a Lamarckian event. However, it may reach the DNA in the form of methylation, acetylation or binding of transcription and cotranscription factors. Similarly, classical experiments in biology also show that embryonic cell development shows a role for environmental borne information flowing towards the cell (8). Like cells tend to associate. Wound healing is an example. Cells dissociated from embryonic chick tissues (same DNA) and injected into the blood stream of host embryos almost invariably come to rest into the proper organ (8). Similarly, when two types of dissociated embryonic cells are mixed in Petri dishes, they will tend to reassociate according to cell-differentiated type and not in accord to DNA sequence that are the same (9). It is important to indicate that in the case of cancer cells, the less differentiated the tumor cell is, the greater are the indications of its lack of growth control. Another side of the same biological form of growth control linked with cell differentiation derives from the simple observation that in normal condition our organs must
have a limit for its growth or its size. This limit must have a link with its functional role while cancer cells growths for its own sake. Cancer cells does not show any cooperative role with the rest of the body as normal cells does through its differentiated cell function. This aspect of cell growth control can be related with the “internal milieu” the extracellular space shared by all differentiated cells maintained under homeostatic control that is absent in microorganisms. Therefore, when microorganisms are used as simpler models for the study of biochemistry of cell differentiation it must be considered that to restrain the discussion on the topics that deal with energetics or maintenance of ATP levels (energy charge (4)) preserve the aspects that are similar on both systems under comparison.
This is what will be here done next. In M. rouxii, part of this harvested free-energy-change from the environment can be found in the amount of carbon from amino acids in the growth media that is accumulated in the form of glycogen. In figure 2, it is possible to see that growing mycelium accumulates glycogen as it growths (low values of mycelium amount/ nutritional media) in black columns. When the amount of mycelium increases beyond certain level, the mycelium growth and glycogen accumulation abilities decrease. If protein synthesis (and growth) is inhibited by cycloheximide the carbon flow from amino acids to glycogen increases in favourable growth conditions. However, at and above 0.98 g of mycelium / 100 ml of media, there is a sharp decrease in glycogen accumulation and in fact, a strong decrease in the glycogen content is observed that does not requires new proteins synthesis in order to be elicited (No DNA through RNA to protein information flow). Therefore, constraining the carbon flow to almost a single macromolecule (glycogen) brings two major experimental aspects to be considered (10, 11) In these references (10,11) “homeostatic” considerations were made by other authors: AThe crowding effect observed on mycelium glycogen levels is amplified by the restricted flow of carbon caused by cycloheximide inhibition of protein synthesis. BThe work is done with a fix amount of mycelium mass throughout the experiment by the action of cycloheximide since no mycelium grow is allowed to occur, even when the crowding effect is not present or in better words when a low relationship of mycelium mass/ volume of growth media is maintained. Furthermore, since mitochondrial function is required in order to have amino acid carbon converted into glycogen carbon, the positive change in glycogen content act as a positive indirect indicator of mitochondrial function. Complementarily, glycogen content decrease indicates lack of mitochondrial function that is replaced by a fast glycogenolysis plus glycolysis required in order to convert the chemical energy of internal glycogen into ATP during a limited period of time. This time limit derives from the fact that for M. rouxii as well as for our cells, life or anaerobic growth and metabolism cannot be sustained in absence of (glycogen) or absence of external source of glucose in growth media (in gluconeogenic condition).
Figure 2
The experiment shown in Table 1 could only be carried out at constant pressure of oxygen (With mycelia placed at that same culture medium at same time at high and low concentrations of mycelium per volume of culture medium) with the use of equipment developed by Prof. José Venâncio Pereira Leite adapted for this test (12). The experiment was carried out in at least duplicate in each condition of the mycelium (high or low cell density) in direct contact with the oxygen supply tube. Three completely independent experiments were performed. The oxygen injection tube was always placed at the outer portion of the culture medium (external to the stainless steel mesh) to prevent increased pressure led by the increased resistance to oxygen flow in the central part of the assembly that could negatively affect, the signal that starts the oxygen generator. Outside this stainless steel mesh, the mycelium could be in high-density or low-density mycelium/volume of culture medium. The medium had a previously sterilized electromagnetic stirrer that drives the media in the same direction of flow of oxygen inlet that keeps a constant supply of this electron acceptor. With the drug cycloheximide, the total mycelium mass was kept constant during the test even though the high and low density portions were switched position (external or internal to the compartment separated by stainless steel mesh from the outside of the culture medium).
Attempts to deliver oxygen directly to the inside of the stainless steel mesh basket failed by uncontrolled system pressure. Each test had a total duration of 1 hour with the two sections of 30-minute each (in some with reversed condition in terms of high low cell density). A total volume of 1.1 liters of culture medium will have within the stainless steel mesh a volume of approx. 350 ml and 750 ml outside. In a typical experiment, 11.11 g of mycelium obtained after 18 hours of aerobic cultivation under gluconeogenic condition were placed outside the stainless steel mesh in a volume of approx. 0,75 L or placed in total 1.1 L (1.1%) with no initial respiration. After separating low-density 3.58 g / 750 ml (0.47%) from high-density condition 6.17 g / 350 ml (1.76 g%) within the stainless steel mesh respiration reached 59 microliters O2 / mg . hour as shown in table 1 in the respiring sample. The respiring sample is obviously the one that increases its glycogen content from the original 3,1 to 5,0 mg/g of mycelium. While in the high-density condition in the same culture medium, the glycogen levels were reduced from the same 3,9 to 1,8 mg/g of mycelium. The system of oxygen consumption measurement under maintenance of constant oxygen pressure is described in detail for use in rodents and small mammals (12). However, was also adapted for small children and for small samples as from bovine semen stored for use in artificial insemination. The system uses a circulating air system with removal of carbon dioxide and water vapor and is based on the electrochemical production of oxygen that is triggered as soon as there is a pressure drop at constant temperature. His oxygen production capacity can be adjusted according to the desired purpose and the equipment used for M. rouxii measurements could work with two oxygen generation capacities: at 100 mA or 250 mA of current. In the first case, we would have 6 Coulombs per minute, and in the second case 15 coulombs per minute. The first would generate 0.49741 mg (348.19 microliters) of oxygen per minute and the second; 1.2435 mg (870.45 microliters) of oxygen generated by the system to replace the oxygen consumed thus keeping constant its offer during the assay. For standard conditions of temperature and pressure, the time ratio (TO/TT) between the machine producing oxygen (TO) and total time it remaining circling the air in the camera (TT) allows measurement of oxygen consumption using the following relation TO /TT x 348.14 = microliters of oxygen consumed per minute in Standard conditions in the first setting. For the second setting: TO / TT x 870.45 = microliters of oxygen consumed per minute, while the partial pressure of oxygen was held at constant value. This final aspect above mentioned, constant oxygen supply, is important for the following reason; Same genome, same metabolomics in the nutritional media and differential interaction mostly likely do to the fact that flow is not an scalar measurement among other aspects of proteins as for instance, interactional agents in oscillatory systems that will not be here discussed. It is important to indicate here that, in organs submitted to complete ischemia oxygen consume stops when still there is enough oxyhemoglobin available (13, 14)
3- Cell differentiation, growth control metabolic pattern under internal or external regulation (homeostasis)
Our mature red blood cells as already mentioned depends upon anaerobic glucose metabolism during its entire life spam. Other cells types of mammals must depend upon transient periods of anaerobic metabolism. These observations also indicate the importance of blood glucose homeostatic control for life maintenance. In addition to that, these observations call attention to another aspect of anaerobic glucose metabolism, for some cells during defined period there is no metabolic pathway that can function as a replacement for this source o ATP. Any cell that does not receive enough oxygen for a period of time will rely on anaerobic metabolism as its sole metabolic pathway for preserving life even when no differentiated cellular function can be preserved at the same time. When this observation takes into account the natural history of almost all differentiated cell found in mammals it is clearly established a metabolic link with previous dedifferentiated state or at least less differentiated state of any cell line. Therefore, at least for a period of time this cell will be receiving from its environment a signal that indicates that its metabolism must function in condition similar to the ones found in foetal or in its precursor stem cell line. M. rouxii presents an example of differentiation that does not have perfect equivalence with the one found in mammalian cells. For instance, while for the mammalian cells the external medium have some components whose values are kept inside a normal range of values, thanks to the work of other differentiated cells ( through homeostatic control absent in microorganisms) in microorganisms the environmental changes may affect metabolism and in the case o M. rouxii morphogenesis. In addition, the stability of this extracellular “internal milieu” requires complex function of integrating systems (neural, hormonal and the like that are absent in microorganisms). In M. rouxii is the change in its extracellular condition (in the environment) that is the key for its differentiation. Furthermore, some of its morphological states depend upon the presence of for instance, glucose in the nutritional media to allow for its metabolism (in yeast-like form for instance since, anaerobic glycolysis is its major source of ATP). However, with some care in the analyses of the results there is a clear relationship between metabolism and differentiated morphology. Therefore, aerobic metabolism, mitochondrial function, and wide range of nutritional sources of carbon allowing for its growth and a sharp limit of growth related the crowding effect are all related to mycelium growth. On the other side, yeast like morphology, absence of mitochondrial function, dependence of glucose in the nutritional media, lack of the same limit of growth found in aerobic condition by crowding effect seems to compose another set of nutritional and metabolism associated with yeast like cell morphology. In very general terms, M. rouxii present morphogenetic form of Pasteur Effect since in anaerobiosis we have yeast-like morphology and in aerobiosis mycelium morphology. This pattern of M. rouxii linkage between metabolism and morphology has led to several research lines. For instance, Phenetyl alcohol repressing mitochondrial function and leading to yeast-like morphology in presence of air (15). In this case, as the crowding effect was ignored at that time a persistent effect of phenetyl alcohol was described since mold morphology did not developed in high density aerobic culture after the removal of the mitochondrial inhibitory drug (Phenetyl alcohol). On the other hand, (shortly,after our first work in M. rouxii Pyruvate Kinase, Terenzi moved from “Instituto de Investigaciones bioquímicas” in 1972 to “Facultad de Farmacia v Bioquimica, Departamento de Ciencias Biológicas, Universidad de Buenos Aires, Argentina” despite a continued collaboration with H.
N. Torres). Terenzi, in the new group, was able to show, early in 1973, that possible electron acceptors EDTA (+/- Zn) (16) alone or in metallic complex form, as previously found with methylene blue, allowed for mycelium development under anaerobiotic condition. Most likely, through these drugs functioning as partial replacement of oxygen for mitochondrial function. Interestingly, the same work also shows that reduced mitochondrial function in M. rouxii was accompanied by increased pyruvate kinase activity (16). In addition, in absence of Mg a divalent cation strongly linked with the function of proteins involved in ATP use or synthesis led to abnormal morphogenesis. In much resumed form, a normal mycelium development requires for the tip growth pattern of hyphal elongation a functioning mitochondria In the case of the liver, as already mentioned, Claude Bernard found it to be a key regulator of blood glucose, fast metabolic regulation are very much more complex. Our previous research about hepatocytes of liver under extra-hepatic obstruction a condition that is not so severe for these liver cells as toxic or viral hepatitis can be, have shown that mitochondrial ATP generating function decreases as long as the obstruction period of time increases as was previously found by others (17, 18). Latter, the Chlorpromazine effect was shown to be, in general terms similar to the one found for other researchers in the making of ischemia a reversible event, as long as the drug is administered previously to the ischemic process (19, 20). Following this line of reasoning a progressive reduction in portal blood flow was found in extra hepatic cholestasis and the flow increase immediately after biliary drainage also indicates the link between increased hydrostatic pressure in bile duct system and reduced portal blood flow to the liver (partial and/or functional ischemia (18, 21). Finally, the pattern of increased pyruvate kinase activity has completed as a reinforcement of the idea that extra-hepatic cholestasis led to ischemic process that could explain through reperfusion injury some unexpected results of this surgery. Finally, the lack of hormonal response of this enzyme was found to be independent of any effect of blood-increased metabolites derived from cholestasis (21). Therefore, the great changes in liver metabolism that were good predictors of surgical outcome could be linked to the Pasteur Effect pattern of metabolic response (17-22). This is a clear case where liver differentiated function is prevented by metabolic condition in liver living cells. Even when placed chemical replacements of hormonal signals that would work inside the liver cell the protein pyruvate kinase in its active R conformation could not be inhibited by what mimics hormonally activated Pyruvate kinase phosphorylation. This result, clearly shows that with the same amino acid sequence the enzyme could be in a conformation that favours the reception of the hormonal signal or alternatively in a conformation that blocks the arrival of the hormonal signal. The inhibitory hormonal signal, is essential for the inhibition of pyruvate kinase that allows for activation of the opposite pathway (gluconeogenesis). However, under a condition of decreased mitochondrial function, pyruvate kinase is the sole producer of ATP available to replace mitochondrial function. Therefore, it can´t be inhibited without leading to cell death. In this case, life in the cell is preserved while differentiated cellular function (neoglycogenetic activity) is blocked. Therefore, it is possible to perceive that to be alive is a cell requirement for differentiated cell function. However, as shown in cardioplegia also, and here in liver function during extra hepatic cholestasis, it is clear that to be alive is not enough for a complete display of cell differentiated function. In medical induced cardioplegia, increased potassium blood levels under medical control stops heart cell differentiated function without killing heart cells or damaging its genome. In extra hepatic cholestasis, decreased portal blood flow to the liver leads to increased pyruvate kinase activity to temporarily maintain intracellular ATP levels while at the same time, this conformational change in the enzyme prevents liver gluconeogenic metabolic pathway, a differentiated liver
cell function that is required to maintain extracellular blood glucose levels. Take into account that cell differentiated function seems to be linked with cell growth control in mammals and with structure of membrane lipids that are not coded for in the genome. It is clear that other cells may find a form to be alive without performing any the differentiated function that is required for the homeostatic chemical pattern of the extracellular shared with other cell types, tissues and organs. Since the internal condition of some cells, due to external factors, may allow to be alive only and not to fully differentiate. In M. rouxii, can environmental condition of living cells prevent the display of biochemical activities required for morphogenesis? Regarding this question, advantage was taken from the fact that yeast like cells growths in presence of glucose beyond the level of crowding effect of mycelium (0,98 g%). Therefore, the conversion from anaerobic growth to aerobic one could be attempted beyond the crowding limit. The result can be seen in Figure 3 when abnormal morphogenesis could be found when yeast like cells are placed under aerobic condition above the cell density that prevents mitochondrial respiratory activity. This conflicting environmental signals presence of oxygen beyond plus the cell density control of mitochondrial activity shows that even in this microorganisms a condition that preserves life may not allow for a complete morphogenetic development and a cell may be held alive in a partial functional condition. That life is preserved can be perceived by the fact that simple dilution of mycelium in growth media instantaneously led to normal mycelium development. The same reversion of normal development after mycelium dilution in normal growth medium was obtained for yeast-like cells showing “persistent effect” of phenetyl alcohol (15). An effect that was seen even after removal of phenetyl alcohol after long period of growth in the presence of the drug when higher than normal amounts of yeast like cells were found in the culture medium (g% above 0,98). In this case if the yeast were harvested, washed and diluted in the same amount of culture media (above 0,98g%) they were previously in, even without the drug, the yeast-like morphology previously induced by the drug in aerobiosis (lack of mitochondrial function) persists. It is fair to assume that stem cells of mammals could be placed in its niches in mid/partial anoxic condition with mixed signals requesting differentiated function of more mature cells while favouring expression of foetal (anaerobic) genes. In this condition, metabolism moves to low yield ATP production mode, high carbon flow on anaerobic glycolysis pathway and insufficient ATP for quality control of fast duplicating DNA. In this condition, mutations may arise from normal genomes without being corrected by the usual quality control mechanisms. Auto assembly keeps this undifferentiated high replicative cells alive that do not show any kind of control by those mechanisms linked with the general whole body control of differentiated cell function. Therefore, in good health conditions, both regulatory targets, intracellular and extracellular, must count on auto assembly among other factors, in order to allow for some preconditions required for those two regulatory domains be kept in regulatory harmony. However, besides genetic factors, environmental ones may have a role in the development of a deregulated undifferentiated living form as cancer cells are. Since the late seventies early eighties molecular biology reasoning predominance has led DNA, to take a central role in biochemistry, the sole biological regulation must include a role for transcription factors. Proteins inside the cell were considered as static elements as its amino
acid sequences are. Therefore, any regulatory mechanism that did not include a need for genetic expression change became part of a fossil form of reasoning that should not be taken into account. For this reason, among others, the work presented in (10) took a long period of time and effort in order to be published (11) and the work that was ready in the following year of (10) presentation (23) was discontinued. The result of the change of focus from biochemistry to genetics (molecular genetics) and from proteins to nucleic acids chemistry was, an increased number of medical errors that are clearly linked with deterministic reasoning in various forms. Conformation of proteins was clearly what E. Schrodinger considered involved in normal distribution of measurements, while the coding capability of the Chromosome fiber (obviously in his opinion, the DNA) the stable and deterministic component of genetics in his seminal book: What is Life?. Biochemistry could provide the molecular reasoning for physiology and genetics in the future of his time. For very complex reasons that will not be addressed here in detail, only the genetics leg grew and in the reverse from the detail to dogmatic generalizations. A complete blockade of the normal biochemical reasoning that historically considered proteins as interaction macromolecules has occurred. While coding macromolecules were considered as non-interacting ones in order to preserve its stored inheritable information. Fast regulatory mechanisms no longer exists since all regulation must involve transcription factors. There is no such a thing as “regulatory genes”. There are genes that code for regulatory RNA and/or proteins. The result is clear: 1- The tragedy of mad cow disease (24). Because an ingested protein is forbidden of a plausible transfer of its wrong conformation to an internal recently made protein. Despite of the fact that allosteric model was based upon concerted change in protein conformation guided among subunits. 2-The difficulties to acknowledge that a phenomenon well described in a Biochemistry book of the early eighties explained the reason for increased death rate among humans that showed decreased blood potassium levels in a clinical assay of a drug made in various well developed countries (25). 4- Concluding Remarks Proteins are macromolecules that have being baptised according to a deity that changed its form as soon as was observed. This characteristic of conformational change remained for quite a long time as its best-known feature; several biological events that require change in protein conformation in order to be explained in molecular terms can be listed from the 1950 onwards. Therefore, when technological advances led to measurements of this very fast events (protein conformational change) on several levels of speed (from femtoseconds to minutes) it came as no surprise that to some stent these measurements must be kept hidden under a curious title for the excellent revision (26). Proteins do not change its conformation only its “personalities” (26). Proteins that are capable of changing other proteins or have its conformation changed by other protein must be labelled as “rogue” proteins. How much time is necessary for a “renaissance” of sound scientific reasoning in biochemistry and above all in medicine? Life is not a product of information. . It requires information used under strictly
FIGURE 3
In this photo (previously presented in SAIB XIV 1978 (23)), an abnormal form of development that does not impedes life of M. rouxii is shown. However, this growth condition blocks normal mycelium development in reversible way. In case this culture is diluted, it rapidly develops into normal mycelium forms without these large round cells that seems to be receiving limited condition for mitochondria function similar to the case of cholestatic liver and therefore cannot display fully differentiated function (Microphotography kindly made by Prof Heni Sawaya).
regulated condition as is the use of matter, energy. Minimal genomes in some microorganisms code for the amino acid order of all the proteins required to produce all he building-blocks of its proteins - all the amino acids. On the other hand, the metabolic order of carbon inflow follows a temporary order of source of carbon, one at the time, therefore through this order in time, microorganisms can regulate its carbon flow. Our genome has plenty of room for the coding of all these proteins that microorganisms have. However, our cells use all the sort of organic molecules found in our blood (glucose, amino acids, free fatty acids at the same time). Our genome do not hold all the information required in order to sequentially assemble all the amino acids of the proteins required to produce all the amino acids from all the carbon sources available at the same time. Most likely, the reason for this limit in information derives from the fact that it will be impossible to have a regulation required to preserve life in case, all this information was available in our genome. Carbon flow in growing microorganism follows almost a single direction in order to be excreted or incorporated to a new microorganism. In our system, even during growing periods, there is a large recycling activity that prevents interruption of growth by lack of one or two rare essential amino acids and/or essential fatty acids. It is a price paid for not having all the information in our genome. However, it is a smaller price than the alternative, to have always a “perfect” nutrition. Taking into account, general aspects of biology shared by all known living beings as “energy charge, for instance, it is fair to assume that life is derived from a common ancestor. However, considering the differences in regulatory strategy it is also fair to acknowledge that microorganisms are not our ancestors. Complex animals and microorganisms must have evolved following very divergent pathway from any common ancestors they may have. Regulation cannot be perceived in sequence or in metabolomics alone. It is an interactive process dependent of protein conformation that is always changing itself at least, for regulatory proteins. Similarly, science is not the product of data only; it requires scientific understanding of the data following a safe way that goes from the general references towards the molecular details and not by following the opposite way. References 1 – Bernard, C. 1878 Leçons sur les phenomènes de la vie. Paris: Baillière. 1878.
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