Pasteur Effect – Warburg Effect – What its history teach us today.

José Eduardo de Salles Roselino

References presented by numbers only may be found in two previous texts concerning

Warburg – Pasteur Effect.

1- https://www.academia.edu/12104859/Warburg_Effect_or_Pasteur_Effect_revisited_

with_biochemical_and_biological_links_to_cancer

2- https://www.academia.edu/12658720/II_-

_Warburg_Effect_or_Pasteur_Effect_revisited_-

biochemical_and_biological_links_to_cancer

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic

regulation described. A decrease in the carbon flux originated at the sugar molecule towards

the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells

were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s

studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the

yeast growth media observed after a measured period of time. The decrease of the sugar

concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen

deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic

condition. This finding was very important for the wine industry of France in Pasteur’s time,

since most of the undesirable outcomes in the industrial use of yeast were perceived when

yeasts cells took a very long time to create, a rather selective anaerobic condition. This

selective culture media was characterized by the higher carbon dioxide levels produced by fast

growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results

indicating that chemical and biological oxidation of sugars produced the same calorimetric

(heat generation) results. This observation requires a control mechanism (metabolic

regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative

processes (metabolism). In addition, Lavoisier´s results were the first indications that both

processes happened inside similar thermodynamics limits. In much resumed form, these

observations indicate the major reasons that led Warburg to test failure in control mechanisms

in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million

years that included volcanic activity, tectonic movements of the earth crust, and glaciation,

and more recently the use of carbon fuels and the extensive deforestation of our land masses

have had a large role in determining the biological speciation over time, in sea and on land.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all

classical thermodynamics must be measured in reversible thermodynamic conditions. In an

isolated system, increase in P (pressure) leads to a decrease in V (volume), all this occurring in

a condition in which infinitesimal changes in one affects in the same way the other, a

continuum response. Not even a quantic amount of energy will stand beyond those

parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In

biochemistry, reversible usually indicates a reaction that easily goes either from A to B or from

B to A. For instance, when it was required to search for an anti-ischemic effect of

Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate

system of increased biliary system pressure in a reversible manner to exclude a direct effect of

this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res

1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in

ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken

into consideration since, after several mutations (not affecting previous activity and function),

a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an

example, consider a Prion C from lamb getting similar to bovine Prion C while preserving its

normal role in the lamb and finally, when its ability to change Human Prion C is considered as

equivalent to a new function (Prussiner,S.).

This observation is good enough, to confirm one of the most important contributions of Erwin

Schrodinger in his “What is Life”:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to

an audience of about four hundred which did not substantially dwindle, though warned at the

outset that the subject matter was a difficult one and that the lectures could not be termed

popular, even though the physicist’s most dreaded weapon, mathematical deduction, would

hardly be utilized. The reason for this was not that the subject was simple enough to be

explained without mathematics, but rather that it was much too involved to be fully accessible

to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its

followers described its requirement for aerobic catabolism two major lines of research started

the search for the understanding of the mechanism of energy transfer that explains how ADP is

converted into ATP. One followed the organic chemistry line of reasoning and therefore,

searched for a mechanism that could explain how the breakdown of carbon-carbon link could

have its energy transferred to ATP synthesis. One of the major leaders of this research line was

B. Chance. He took into account that relatively earlier in the series of Krebs cycle reactions,

two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl

carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not

important whether the released carbons were or were not exactly those originated from

glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary

that could act as an energy reservoir. The intermediary could store in a phosphorylated amino

acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its

phosphate group to ADP producing ATP. A key intermediate involved in the transfer was

identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz

Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second

line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical

potential energy in transmembrane ionic asymmetries. They also presented the explanation

for the change from resting to action potential in excitable cells. This second line of research,

under the leadership of Peter Mitchell postulated a mechanism for the transfer of

oxide/reductive power of organic molecules oxidation through electron transfer as the key for

the energetic transfer mechanism required for ATP synthesis.

This diverted the attention from high-energy (~P) phosphate bond to the transfer of electrons.

Most of the time, during the harsh period of the two confronting points of view, Paul Boyer

and followers attempted to act as a conciliatory third party. According to personal accounts (in

L. A. or Latin America) heard from those few of our scientists who were able to follow the

major scientific events held in USA, without getting good results. Afterwards, Paul Boyer could

present how the energy was transduced by a molecular machine that changes in conformation

in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite

direction in order to produce ADP plus Pi from ATP (reversibility). He also received a Nobel

Prize for his work on this matter.

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute,

over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the

cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow,

this outcome represents such a blow to Chance’s previous work that somehow it seems to

have cast a shadow over very important findings obtained during his earlier career that should

not be affected by one or another form of energy transfer mechanism. For instance, Britton

Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and

with the use of this technique he was able to present the idea of control of energy metabolism

(respiratory control rate) that brings us back to Pasteur´s findings.

This metabolic regulation mechanism seems to have been neglected in the recent years of

obesity epidemics, which led to a search for a single molecular mechanism required for the

understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does

not mean that here the role of central nervous system is neglected. In short, in respiring

mitochondria the rate of electron transport linked to the rate of ATP production is determined

primarily by the relative concentrations of ADP, ATP and phosphate in the external media

(cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when

the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the

oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene

expression), throttled down to the resting state. The dependence of respiratory rate on ADP

concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low

respiratory rate. When skeletal muscle is stressed by high exertion, lactic acid produced is

released into the circulation and can be metabolized aerobically by the heart to CO2 or by the

liver that could produce glucose out of two lactic acid molecules.

This respiratory control of metabolism will lead to preservation of body carbon reserves and in

case of high caloric intake in a diet, also shows increase in fat reserves essential for our

biological ancestors survival (Today for our obesity epidemics). No matter how important this

observation is, it is only one focal point of metabolic control. We cannot reduce the problem of

obesity to the existence of metabolic control. There are numerous other factors but on the

other hand, we cannot neglect or remove this vital process in order to correct obesity.

Furthermore, we cannot explain obesity ignoring this metabolic control. This topic is so

neglected in modern times that we cannot follow major research lines of the past that were

interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic

vision of biology could replace all previous knowledge by a new one based upon ATGC

readings. For instance, in order to display bad consequences derived from the ignorance of

these old scientific facts, we can take into account, for instance, how ion movements across

membranes affects membrane protein conformation and therefore contradicts the wrong

central dogma of molecular biology. This change in protein conformation (with unchanged

amino acid sequence) and/or the lack of change in protein conformation is linked to the

factors that affect vital processes as the heart beat. This modern ignorance could also explain

some major pitfalls seen in new drugs clinical trials and in a small or personal scale on bad

medical practices.

The work of both, B Chance and of P Mitchell have deep and sound scientific roots that were

made with excellent scientific techniques, supported by excellent scientific reasoning and that

were produced in a large series of very important intermediary scientific results. Their sole

difference was to aim at very different scientific explanations as their goals (They have

different Teleology in their minds made by their previous experiences). When, with the use of

mutants obtained in microorganisms, P Mitchell´s goal was found to survive and B Chance to

succumb to the experimental evidence, all those excellent findings of B Chance and followers

were directed to the dustbin of scientific history as an example of lack of scientific

consideration. It should also be stressed that while on the one hand, the Mitchell model used

a unicellular microorganisms; on the other, Chance’s work was with eukaryotic cells, quite

relevant to the discussion.

We can resume the challenge faced by these two great scientists in the following form: The

first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up

with the name of Fritz Lipmann. Its central feature was the recognition that adenosine

triphosphate, ATP, serves as a universal energy “currency” much as money serves as economic

currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In

microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason

that we cannot consider that gene expression is equivalent to protein function (an acceptable

error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge

error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and

the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to

presume that to find out how the flow of metabolism (carbon flow) led to ATP production

must be considered a major focal point of research of the two contenders. Consequently, what

could be a minor fall of one of the contenders, in case we take into account all that was found

during their entire life of research, the real failure in B Chance’s final goal was amplified far

beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a

very sound root. Metabolism may produce two forms of energy currency (I personally don´t

like this expression*) and I use it here because it was used by both groups in order to express

their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas1:

The second kind of energy currency is the current of ions passing from one side of a membrane

to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley,

Huxley & Huxley, Huxley & Simmons(1940s to 1972) as a solid support. B. Chance had the

enzymologists involved in clarifying how ATP could be produced directly from NADH + H+

oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate

intermediary. Both competitors had their work supported by different but, sound scientific

roots and have produced very important scientific results while trying to present their

hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance

followers was to create a conflict between what would be expected by a restrictive role of

proteins through its specificity ionic interactions and the general ability of ionic asymmetries

that could be associated with mitochondrial ATP production. Chemical catalyzed protein

activities do not have perfect specificity but an outstanding degree of selective interaction was

presented by the lock and key model of enzyme interaction. A large group of outstanding

“mitochondriologists” were able to show ATP synthesis associated with Na, K, Ca…

asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to

display the existence of antiporters that exchange X for hydrogen as the final common source

of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow

discontinued in the form of scientific research, when the final E. Coli mutant studies presented

the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you

will always be better off in case you have a mutant” (Principles of Medical Genetics T D

Gelehrter & F.S. Collins chapter 7, 1990).

This in much resumed form resumes what was the leading reason to convince anyone that it

would be rather safe to search for DNA sequences data that will be produced anyway, instead

1 *ATP is used under the guidance of cell needs and not by its yield as currency are. When

glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is

required to match in time the cellular needs. On the other hand, when it may yield (in

thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar

to an investor absurd choice since it will place greater value of glucose were it yield least

money for its molecular investment.

of doing any research about molecular mechanisms linked with life and is maintenance that is

a far more risky endeavor.

In addition, it is also considered a wise move to work with microorganisms able to offer

mutants at every new experimental a day.

To do so, it is important i) to assume that anything found in E. coli will be valid for the

elephant. ii) Biochemistry has no time, time must be involved mainly as a component of

genetics (generations) and of evolution therefore the presentation of static data as DNA

sequences are, is good enough for any research purpose. Iii) Furthermore, homeostasis, the

regulation of extracellular parameters, conspicuously absent in microorganisms, may be also

mentioned as if some regulatory events found in microorganisms could be understood as

homeostatic events. The same holds for regulation observed in subcellular organelles that

were also called “homeostatic” despite being intracellular.

In a previous text, the role of citrate as the molecule that changes the enzyme phosphofructokinase conformation to make it inhibited by normal ATP levels (1) the rationale of this mechanism was presented as linked to homeostatic function of the liver. It could be also linked with the flow of carbon from carbohydrates to lipids. Here, in case the citrate blockade is less effective than in liver, the complete mechanism of metabolic flow regulation may count on additional regulatory steps further along the glycolytic pathway. For instance, in pyruvate dehydrogenase step that is inhibited by the oxidation of free fatty acids and ketone bodies (Garland, P. B., Newsholme, E. A. & Randle, P.J. (1964) Biochem. J. 93: 665). When this flow of the carbon of glucose molecule through glycolytic pathway suffers inhibition, there is some increase in metabolites inside muscle cell during heavy exercise. Afterwards, during the recovery period, a thermodynamically problematic (in vivo) reversion of pyruvate kinase activity in preserved muscle preparation may be found (Rampazzo Xavier, A et all 2002 Candian Journal of Physiol and Pharmacol 80(2):164-9) . In addition to that, another display of the importance of moving research from general aspects towards molecular details can be perceived also in the research of muscle metabolism. The rate of glucose uptake and metabolism is greatly reduced in the perfused isolated rat heart and in the isolated rat diaphragm by the presence of ketone bodies, free fatty acids in the medium. In the presence of ketone bodies and free fatty acids citrate levels rises four-fold. The effect of free fatty acids is not a direct one since; in its presence, in case the Krebs cycle is inhibited by respiratory chain inhibitors no glycolytic inhibition is observed (Randle, P.J. , Newsholme, P.J., Garland, P.B. (1964) .Biochem. J. 93, 652.; Newsholme, P.J. Randle, P.J. (1964); Randle, P. J., Garland, P. B. ,Hales, C. N. , Newsholme, E. A., Denton, R. M. and Pogson, C. I. (1966) Rec. Progress. Horm. Res. 22, I). These findings led to the understanding of citrate inhibition over the enzyme phosphofructokinase (PFK). PFK is a major regulatory enzyme in glycolysis. The biological meaning of this inhibition of Phosphofructokinase is perceived as previously pointed out by others, taking into account the Pasteur Effector Pasteur/Crabtree Effect (7). Anaerobiosis during a short period of time in muscle, must rely on glycolytic production of ATP through a low yield mechanism per unit of molecule oxidized that therefore, depends upon a high metabolic flow to reach the amount of ATP required by the cell for unit of time. When the muscle during recovery period at rest, uses aerobic glycolytic production of ATP with a 10 times higher yield for ATP, the glucose uptake and glycolytic flow must be reduced in comparison with the flow measured under anaerobiosis. Citrate is the first molecule formed at the Krebs Cycle and moves to cytoplasm where it “informs” Phosphofructokinase that Glycolysis must have its speed reduced since aerobic glycolysis is ten times more efficient in ATP production than anaerobiotic glycolysis. It does so, by making Phosphofructokinase inhibited by normal values of ATP. Therefore, with Citrate as an allosteric information,

produced by a functional Krebs cycle, glycolytic flow can be adjusted without the requirement for great changes in ATP levels (that otherwise would cause great changes in its free energy change value per mole of ATP spent). AMP also can do this type of informative role since, a small decrease in ATP causes greater percent change in AMP levels than those observed in ATP levels. Increase in AMP activates glycogen breakdown and increases at PFK step the glycolytic pathway. This is the biological meaning inside the cell, as previously mentioned (1). In addition to that, looking outside the cell in complex organisms, - (Those that display C. Bernard , W. Cannon homeostatic regulation of blood glucose)- it is also clear that, when glucose utilization by muscles is restricted by Free Fatty Acids utilization in mitochondria, the general logic of biology must not rely in the cell where regulation is taken place alone. The best meaning for this result is to consider that by inhibition of muscle consumption of glucose, glucose is preserved for brain, red blood cells etc. that have no replacement for glucose burning. Since these tissues/cells are almost strictly dependent upon this organic molecule. This pattern of regulation in PFK is observed all over biology with quantitative variation only. Therefore, ATP inhibition of PFK seems to be a very general “out of the regulatory rule” pattern and looking further away from the reaction under control of PFK it is also part of the Pasteur Effect regulatory control. Further away from the reaction, ATP will be the product of the metabolic pathway and no matter whether in the step it is a substrate, along the pathway it will be a product. This exception, (being inhibited by a substrate) is understood as an exception to the general regulatory rule (being inhibited by product=feedback inhibition) when the enzyme is taken alone. In this case, this enzyme is not following the usual product inhibition rule and, this is another aspect that places serious questions about one gene alone evolution. Here, another very interesting fact must be also considered regarding biological regulatory phenomena inside the cell. This abnormal pattern of regulation makes the protein PFK to behave as an oscillator, a pendulum, a device form of measuring time, something that seems to be a requirement for brain function. Also, under the light of biological evolution, it is reasonable to assume, that calcium jointly stimulation of muscle contraction and glycogen breakdown makes sense. Consider that the fast transition from resting to working muscle may require a quasi-autonomous chemical-metabolic response of muscles in order to face flight or fight emergencies (something absent in liver transition from fed to fasted state a gradual, slow, change). Glycogen breakdown provides an ATP source that can be taken as a rather independent adjustment of intracellular metabolism when the rather limited adjusted blood low for this emergency is taken into account. However, in case the previously mentioned muscle contractions were required for a simple and slow-moving walking, the emergency status must be shut down in the muscle. This is most likely done by inhibition through plenty ATP production under aerobic condition (another aspect of Pasteur Effect) acting over a changed in its conformation - phosphofructokinase. This phosphofructokinase conformational change is prompted by citrate. In case, this glycolytic inhibition is considered as an example of intracellular energetic regulation (equal to the ones observed in microorganisms) it does not make sense. Consider for a while that, the “currency value” of ATP is the same, no matter it is produced by ketone bodies, ketogenic amino acids, free fatty acids or pyruvate derived from glucose in Krebs cycle-respiratory chain of mitochondria. Only when, maintenance of blood glucose levels are taken into account as a result of differentiated cell regulatory function activity, this sparing effect upon glucose metabolism can be clearly understood. Intracellular regulation and extracellular regulation are not reduced to ATP maintenance and

homeostatic blood glucose maintenance. However, for didactic purposes these two regulatory

goals may be considered as the best examples of intracellular and homeostatic targets for

regulation. In this regard, it is wise to assume that the slow moving transition from fed to

fasting in liver is done in a way that accomplishes its goal of blood glucose maintenance with

harmony of regulation for its ATP levels. First already available Glucose 6 phosphate are used,

followed by glucose from Glycogen stored and only latter, the ATP expensive, neoglucogenetic

pathway is started. In the muscle, conversely, fast transition from resting state to full workload

is made following an opposite logic. Initially, an emergency pattern of response is set into

action with very low yield for ATP used as currency. During this initial period of time, the

muscle counts on a low yield source of ATP that is also, an independent form of energy source

for muscle work (fast glycogen breakdown observed by Leloir in 1968). Only latter, if not in an

emergency condition, therefore not requiring an independent source of energy, a high yield

form of ATP production is set and during this condition, a switch from glucose to others

sources of ATP (all aerobic ones) contributes to the whole body harmony through the

preservation of blood glucose levels. These regulatory adjustments are made in time, time is

conspicuously absent from ATGC sequences alone. They use Pasteur Effect mechanisms for

ATP maintenance and require a very complex level of auto organization in order to avoid futile

fluxes of equal level of opposing metabolic pathways inside the same cell where ATP must be

preserved. In case muscle and liver metabolism is regarded, the same rule of ATP preservation

is not required and the whole body works fine with lactate production in the muscle occurring

at the same speed of lactate conversion into blood glucose in the liver, as long as free fatty

acids provides the required source of ATP for liver gluconeogenesis. It is now very clear that

the differences between the two regulatory domains presented by intracellular and the

extracellular where homeostasis occurs is observed. The last one is quite different from the

one found in microorganisms and the intracellular found in homeostatic beings must be kept in

harmony with its extracellular regulatory mechanisms therefore, despite bearing resemblances

with regulation found in microorganisms also have some peculiar aspects of their own.

In resume, Pasteur Effect was first observed in microorganisms and still can be studied in these

simpler systems. In mammals, Pasteur Effect preserves some general aspects linked with the

transition of aerobic/anaerobic metabolism and its strong link with carbohydrate metabolism

as was observed by Pasteur in yeasts but may require a detailed knowledge of the

differentiated function of the cell, tissue, organ or system where it is being observed in order

to grasp its real evolutionary meaning. In this last aspect, short time available between

environmentally born event (signal input) and its biological regulatory response (regulatory

output) for biochemical responses, places limits for mechanisms that can be included in the

regulatory role for life preservation. For instance, this limit in time excludes mechanisms

dependent upon changes in gene expression despite of the fact that they may have as

intermediary steps, continuing the input signal, through fast changes in protein conformation

among some transcription factors. During these intermediary steps of biological adaptation to

environmental changes, the signal is still moving towards the DNA but without causing any

change in triplet order (genetic information). Change in triplet order would be something that

in case it happens will give support to wrong Lamarckian view of evolution. Also, during this

early period of biological response to some environmental change the biochemical response

occurs without any change in gene expression, something that will come latter. Only

biochemistry, through biochemical regulation, mainly due to changes in protein conformation

is preserving life.

As pointed out by Larry Bernstein:

“Glycolysis is enhanced and beneficial to cancer cells. The mammalian target of rapamycin

(mTOR) has been well discussed in its role to promote glycolysis; recent literature has revealed

some new mechanisms of how glycolysis is promoted during skin cancer development.

On the other hand, Akt is not only involved in the regulation of mitochondrial metabolism in

skin cancer but also of glycolysis. Activation of Akt has been found to phosphorylate FoxO3a, a

downstream transcription factor of Akt, which promotes glycolysis by inhibiting apoptosis in

melanoma. In addition, activated Akt is also associated with stabilized c-Myc and activation of

mTOR, which both increase glycolysis for cancer cells.

Nevertheless, ras mutational activation prevails in skin cancer. Oncogenic ras induces

glycolysis. In human squamous cell carcinoma, the c-Jun NH(2)-terminal Kinase (JNK) is

activated as a mediator of ras signaling, and is essential for ras-induced glycolysis, since

pharmacological inhibitors if JNK suppress glycolysis. CD147/basigin, a member of the

immunoglobulin superfamily, is high expressed in melanoma and other cancers.

Glyoxalase I (GLO1) is a ubiquitous cellular defense enzyme involved in the detoxification of

methylglyoxal, a cytotoxic byproduct of glycolysis. In human melanoma tissue, GLO1 is

upregulated at both the mRNA and protein levels.

Knockdown of GLO1 sensitizes A375 and G361 human metastatic melanoma cells to apoptosis.

The transcription factor HIF-1 upregulates a number of genes in low oxygen conditions

including glycolytic enzymes, which promotes ATP synthesis in an oxygen independent manner.

Studies have demonstrated that hypoxia induces HIF-1 overexpression and its transcriptional

activity increases in parallel with the progression of many tumor types. A recent study

demonstrated that in malignant melanoma cells, HIF-1 is upregulated, leading to elevated

expression of Pyruvate Dehydrogenase Kinase 1 (PDK1), and downregulated mitochondrial

oxygen consumption.

The M2 isoform of Pyruvate Kinase (PKM2), which is required for catalyzing the final step of

common aerobic/anaerobic glycolysis, is highly expressed in cancer cells; whereas the M1

isoform (PKM1) is expressed in normal cells. Studies using the skin cell promotion model (JB6

cells) demonstrated that PKM2 is activated whereas PKM1 is inactivated upon tumor promoter

treatment. Acute increases in ROS inhibited PKM2 through oxidation of Cys358 in human lung

cancer cells. The levels of ROS and stage of tumor development may be pivotal for the role of

PKM2.

Harmonic regulation of intra and extracellular is not granted by something that can be

described as a “normal genome” alone. In cells bearing “normal genomes” the transition of

signals that drive biochemical reaction to maintenance of ATP levels may prevail upon those

biochemical reactions required for homeostatic functions through cell differentiated functions.

This can be perceived in extrahepatic cholestasis lack of neoglucogenic response during

ischemia or anoxia. Similarly, in any non-genetic condition that may lead to abnormal

potassium blood levels its effect upon heart muscle beating can be lethal. “Normal genomes”

does not determine normal protein conformation under all environmental condition or in

other words, whole body normality is not determined by normal amino acid sequence alone.

Muscle function also indicates the relationship of fast regulatory mechanisms for function and

slow ones that requires changes in gene expression and affects muscle size. When nerve

impulses causes muscle contraction after changes in protein conformation and these changes

can be quickly reverted by resting. In case of frequent nervous stimulus for muscle contraction,

a conformational change in titin molecules elicits through changes in factors that affect gene

expression hypertrophy that is not quickly reverted by resting. Complementarily, denervation,

through lack of basal stimulus causes muscle atrophy something that can be also caused by

abnormal levels of muscle resting.

This should be understood by considering that initial stimulus set two processes into motion,

one does not require changes in gene expression and occurs in short period of time while the

other requires permanence in time and further time to display its biological responses. In case

of stimulus during short period of time only the easily reverted biochemical responses

(changes in protein conformation) are transiently shown by the cells. This does not have to be

interpreted as an indication of a requirement for other kind of regulatory input in order to

display less revertible regulatory responses (those that affect gene expression). This

observation shows that fast regulatory response arise in short period of time while adaptive

ones requires larger period of time. When the stimulus is of short duration the initial response

of the slow regulatory mechanism also reverts and the slow biochemical response does not

appears. Fast regulatory responses most frequently are associated with intracellular regulatory

needs or undifferentiated cell function but may also be perceived in differentiated function as

well. In common in both cases these biochemical regulatory output will not require changes in

gene expression.

Slow regulatory response, requires changes in gene expression and also requires life

maintenance fast regulatory response. Usually they are seeing in developmental or adaptive

regulation. When a fast regulatory response (required for life maintenance) cannot be

preserved during adaptive process a mechanism of damaging control may be set into motion

and death may be accelerated, gap junctions between cells closed, something that can be

understood as part of a mechanism developed in order to prevent spreading damage from cell

into tissue damage. This is found in survival of heart ischemia and frequently the damaged

area may become fibrotic non-functional area of the heart. It is also, a very important aspect

of neural/brain normal development mechanism. The opposite link between fast and slow

regulatory mechanism or undifferentiated/differentiated biochemical activity is not a cell

requirement. To be alive a cell does not require a cell differentiated biochemical activity, part

of this set of cells, are the several types of cancer cells. Very adapted for survival and devoid of

cell differentiated function (those functions that display the results of their activities in the

whole body and not in the cell itself). Also SirT and PGC1 may have a similar role for

instance, in liver adaptation to a high protein diet through slow regulatory change that uses

changes in gene expression. During daily (circadian) response to fasting only fast and less

demanding in ATP responses are required. However, in case, of changing diets for a seasonal

period the liver may be adapted to high protein diet with changed gene expression.

However, let us take the example of a mechanical wristwatch. When the watch is working in

an acceptable way, which is its normal functioning condition it is easy to perceive that

“normality” is not the result of one of its isolated components - or something that can be

shown by a reductionist molecular view. Usually it will be considered that it is working in an

acceptable way, in case it is found that its accuracy falls inside a normal functional range, for

instance, one or two standard deviations bellow or above the mean value for normal function,

what depends upon the rigor wisely adopted. While, only when it has a faulty component

(similar to a genetic inborn error) we can indicate a single isolated piece as the cause of its

failure (a reductionist molecular view). Molecular biology have led to a faulty idea that

“normality” could be also found as a result of deterministic and molecular view of biology as

are seeing inborn errors.

We need to teach in medicine; first, the major reasons why the watch works fine (not saying it

is “automatic”). The functions may cross the reversible to irreversible regulatory limit change,

faster than what we can imagine. Latter, when these ideas about normal are held very clear in

the mind set of medical doctors (not medical technicians) we may address the inborn errors

and what we may have learn from it. A modern medical technician may cause admiration

when he uses an “innocent” virus to correct for a faulty gene (a rather impressive

technological advance). However, in case the virus, later shows signals that indicate that it was

not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be

found. Even those oscillatory changes in Ca that were shown to affect gene expression (C. De

Duve) were laid to rest since, they clearly indicate a source of biological information that

despite the fact that it does not change nucleotides order in the DNA, it shows an opposing

flux of biological information against the dogma (DNA to RNA to proteins). Another, line has

shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used

for Ca uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L.

Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to

him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of

a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in

Ca load. As some of Krebs cycle dehydrogenases were activated by Ca, this finding was used to

propose a new control factor in addition to the one of ADP (B. Chance). All this was

discontinued with the wrong use of calculus (today we could indicate bioinformatics in a

similar role) in biochemistry that has established less importance to a mitochondrial role after

comparative Kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic

medical courses that must urgently reverse the faulty trend that tries to impose a view that

goes from the detail towards generalization instead of the correct form that goes from the

general finding well understood towards its molecular details. The view that led to curious

subjects as bioinformatics in medical courses as a training in sequence finding activities can

only be explained by its commercial value. The usual form of scientific thinking respects the

limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as

a form to surpass lack of mathematical equation that defines relationship of variables and the

determination of its functional domains. It also uses old scientific roots, as its sound support

never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of

DNA was found as a technical advance to find amino acid sequence in proteins it was just a

technical advance. This technical advance by no means could be considered a scientific result

presented as an indication that DNA sequences alone have replaced the need to study protein

chemistry, its responses to microenvironmental changes in order to understand its multiple

conformations, changes in activities and function. As E. Schrodinger correctly describes the

chemical structure responsible for the coded form stored of genetic information must have

minimal interaction with its microenvironment in order to endure hundreds and hundreds

years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out

in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent

that no longer could be considered to be an inert bath for catalytic activity of the enzymes

when the transfer of energy include a role for hydrogen transport. The great increase in

understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough

to confirm once more, one of the most important contribution of E. Schrodinger in his What is

Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to

an audience of about four hundred which did not substantially dwindle, though warned at the

outset that the subject matter was a difficult one and that the lectures could not be termed

popular, even though the physicist’s most dreaded weapon, mathematical deduction, would

hardly be utilized. The reason for this was not that the subject was simple enough to be

explained without mathematics, but rather that it was much too involved to be fully accessible

to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work

conformational energy as a property derived from our atomic structure can be neutral,

positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity

measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this

very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it

possible that this knowledge may advance to little just a short of a complete understanding -of

the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have

occurred or have not occurred and yet does not present a proof about how it would have

being occurred. It will always be an indication of possible against highly unlike and never a

scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings

that indicates wrong directions in the future, as was his case, or directed toward our past.