Interrelationship in Organized Biological Systems

Abstract

Ensuring synchronization between processes which make up a complex system requires functional interdependence, with multiple mechanisms cooperating to achieve a predetermined final state. Interrelationship involves multiple systems, each equipped with its own regulatory mechanism and hence operating as a distinct entity. Such structural and functional isolation is evident in many complex systems. Mutual dependencies may take on two forms: cooperation and coordination. If the product of one process acts as a substrate for another, we are dealing with cooperation. In such cases both processes are subject to their own separate control mechanisms. Coordination occurs when one process acts directly upon the control mechanisms of other processes, thereby establishing hierarchical dependence. Biological hierarchies arise due to allosteric properties of receptor units which can register signals issued by other autonomous processes. Compartmentalization of mutually dependent processes optimizes the functioning of cellular machinery.

Interrelationship is a prerequisite of function in organized structures:

• 1. The diversity of cooperation mechanisms in cellular and organic metabolism.

• 2. The diversity of coordination mechanisms in cellular and organic metabolism.

• 3. Hepatic cell activity during periods of energy abundance.

• 4. Hepatic cell activity during periods of energy deficiency.

• 5. Co-action in distress—acute shortage of oxygen or glucose.

• 6. Why do cells contain organelles?

• 7. The properties of regulatory hierarchy (organism vs. cell).

• 8. Coordination of metabolism and the role of biological clocks.

• 9. Pathologies resulting from disruption of co-action (e.g., tumors).

• 10. Glucose levels as the coordinating factor in energy management—properties.

• 11. Why can an anthill be referred to as an organism?

• 12. What are the consequences of glycolysis—a key aspect of energy management—occurring outside of the mitochondrion?

• 13. How is co-action between hepatic and muscle tissues realized?

• 14. How are programs of action encoded in biology?

5.1 The Need of Mutual Relations in Biological Systems

Assuming that all structures involved in biological functions belong to regulatory loops allows us to determine—even considering the limitations of our scientific knowledge—the principles of regulation and mutual relations in such automatic systems (as opposed to non-automatic systems, subjected to external decision-making mechanisms and therefore unpredictable when acting on their own). Automaticity is tied to independence, which can only be achieved in the presence of a negative feedback loop. Individual components of an automatic system cannot be considered independent.

As processes owe their independence to automatic control mechanisms, they need to cooperate with one another to fulfill specific biological goals.

Stabilization of substance concentrations and/or biological activity is a result of genetic programming implemented by the cell. In an ideal organism or cell existing in an unchanging environment, all forms of cooperation other than the exchange of reaction substrates could be neglected (in particular, there would be no need for activation or inhibition mechanisms). In reality, however, interaction with the external environment introduces random stimuli which translate into unpredictable fluctuations in the intensity of biological processes and the availability of key substances. Such fluctuations can be caused by inflow and outflow of substrates and reaction products as well as by forced activation of cells. The problem is compounded by the existence of a barrier (membrane) which hampers transmission of substances and signals between the cell and the interstitial space. Unpredictability becomes particularly troublesome in processes whose individual stages occur in different areas of the cell.

Significant deviations from the norm may arise as a result of variable intensity of biological processes. While oscillations are an inherent feature of systems subjected to automatic regulation with negative feedback loops, they may become a problem if the resulting changes are too severe.

5.2 Cooperation and Coordination

Regulatory systems may assist one another either by cooperation or by coordination.

Cooperation is understood as correlation of independent processes based on a shared pool of products or substrates, where neither process is subordinated to the other. Coordination is also a manifestation of correlated activities, but in this case one process is in direct control of the other, giving rise to a hierarchical structure. Biological cooperation may be compared to a contract between two factories, one of which makes ball bearings, while the other uses them in the machines it assembles. In a cooperative system, both factories retain their independence. If, however, the ball bearing manufacturer were to become a branch of the machine factory or if its production rate was fully dependent on the machine assembly rate, we would be dealing with more than a simple sale/purchase contract. In such circumstances, control over the entire manufacturing process would rest with the machine factory. Clearly, such centralized control introduces a hierarchical structure and should therefore be treated as a form of coordination.

Similar properties can be observed in biological systems. While the “technicalities” of coordination and cooperation mechanisms differ from process to process, their general principles remain the same. Understanding them provides insight into the structure and function of biological systems.

The nature of a negative feedback loop, which underpins biological regulatory systems, is to restore stability. Maintaining a steady state is therefore—out of necessity—the core principle of biological programming, both in the cell and in the organism as a whole.

Major deviations from the evolutionarily conditioned substance or activity levels are caused by uncontrollable, random events originating in the external environment. This applies both to cells and to organisms. Individual cells operate in a state of relative homeostasis, but they also participate in hierarchical systems, and their function is subordinated to the requirements of their host organism. The organism can force activation or deactivation of certain cells in order to stabilize its own vital parameters, such as blood glucose levels. The signals issued by the organism (hormones) are treated as commands and may activate various mechanisms in each cell for the benefit of a distributed process, producing results which the cell would not otherwise generate on its own.

The organism comes into frequent contact with the outside world; for instance, through variations in food intake, however, such interaction presents dangers for the state of homeostasis.

Due to the expected randomness and fluctuations, biological systems cannot rely solely on cooperation. The organism responds to these challenges by coordinating its internal processes via hormonal signals.

In order to perform the action requested by the organism, the cell must first ensure its own stability by means of internal coordination. It does so by generating further signals which guide its internal processes. This property allows the cell to counteract major deviations which emerge as a result of inconsistent activity of internal regulatory mechanisms.

In individual cells, both cooperation and coordination are enhanced by the limited space in which the linked processes need to take place.

Substance exchange is the most basic form of cooperation. As such, it is frequently observed in living cells. Examples include the coupling between DNA synthesis and the pentose cycle (which provides pentoses), between glycogen synthesis and glucose-6-phosphate synthesis, between globin synthesis and heme synthesis, etc. Such cooperation occurs when the product of one reaction is a substrate for another reaction, resulting in a “supply chain” condition. Increased demand for the final product causes a decrease in its concentration. This, in turn, triggers control mechanisms which act to increase supply. Figures 5.1, 5.2, and 5.3 present the cooperation of automatic processes where the product of one process is used as a substrate in the effector unit of another process.

Fig. 5.1

The principle of cooperation between processes subjected to their own negative feedback loops. (a) The product of one process used as a substrate in the effector unit of another process, in accordance with a negative feedback loop. (b) The product of one process used as a substrate for many other processes (e.g., glucose-6-phosphate synthesis providing substrates for the pentose cycle, glycolysis, glycogen synthesis, etc.)

Fig. 5.2

Cooperation as applied to cellular metabolism

Fig. 5.3

Cooperative alignment of the Krebs cycle with other biological processes

If, however, the cooperation of two processes is subject to significant disruptions (for instance, if both processes occur in separate compartments of the cell or if the rapid reaction rate makes storage of intermediate products unfeasible), coordination becomes a necessity. Coordinating signals act as “administrators,” increasing demand for overproduced substances or inhibiting the processes which produce them. Coordination is usually implemented by exploiting the product of one reaction as an allosteric effector (rather than a substrate) in another process. The controlling process modifies the sensitivity of the receptor loop instead of directly altering the rate at which a given product is consumed. Figure 5.4 presents a model view of coordination.

Fig. 5.4

Model view of coordination. The product of one process affects other processes which are also subject to their own, independent negative feedback loops (the coordinating product downregulates the receptor by reducing its affinity to its own product). (a) Coordination of indirectly coupled processes (without direct cooperation) and (b and c) coordination of directly coupled processes (with direct cooperation). Solid lines indicate cooperative links. Dashed (colored) lines indicate coordinating signals

In systems which rely on negative feedback loops, the stabilization curve typically assumes the form of a sinusoid. The effects of cooperation and coordination can be described as changes in its shape or oscillation level.

The action of a cooperating system which consumes a given substance may result in changed frequency and (optionally) amplitude of oscillations of its stabilization curve. Increased demand for the product precipitates a decline in its concentration and triggers an increase in its production rate. However, as the receptors of cooperating systems do not change their sensitivity, the target product concentration must remain the same. Thus, any changes are effectively limited to the shape of the stabilization curve (see Fig. 5.5). On the other hand, coordinating signals (action of allosteric effectors) alter the sensitivity of receptors, thereby modifying the initial biological program and forcing a change in the target product concentration or activity level (Fig. 5.6). This phenomenon manifests itself as a change in the slope gradient on the attached allosteric receptor transformation diagram (Fig. 5.7), similarly to the effect of allosteric effectors on hemoglobin. It can be observed both in the covalent modification of enzymes (i.e., the action of hormones) and as a result of noncovalent interaction initiated by an allosteric effector (e.g., an intracellular coordinating signal).

Fig. 5.5

Change in the frequency of the product stabilization curve in a self-controlled process, resulting from increased activity of a cooperating system (increased demand for the product)—an example of product concentration variability

Fig. 5.6

Change in the target concentration as an effect of coordinating processes (decreased receptor sensitivity results in increased concentration of the product, while an increase in sensitivity causes the product concentration to decrease)

Fig. 5.7

Allosteric receptor transformation curve: changes resulting from the effect of coordinating signals (dashed line) compared with the initial state (solid line). Product concentration increases in order to compensate for the decrease in receptor sensitivity (and vice versa)

In general, coordination can be defined as any action which affects the sensitivity of receptors other than one’s own, facilitating interrelation of separate systems in the pursuit of a common goal.

As both regulatory and coordinating mechanisms exploit the allosteric properties of signal-receptor interactions, distinguishing them may appear difficult. We can, however, assume that any loop in which the detector subunit exhibits affinity to the product released by its corresponding effector is a regulatory mechanism. If the detector also registers signals from other systems, the changes triggered by such signals may be counted among coordinating effects (Figs. 5.8 and 5.9). For instance, if a blood glucose concentration receptor triggers a process which results in modification of blood glucose levels, the action of the system can be best described as self-regulatory. If, however, the same detector also activates other processes, resulting, e.g., in modifying the concentration of fatty acids in blood, then we are dealing with coordination.

Fig. 5.8

Control (regulation, colored line) of coupled processes via cooperation (solid line) and coordination (dashed line), as applied to amino acid synthesis. Each fork in the synthesis pathway involves separate coordinating mechanisms. Symbols are explained in the inset. Symbolic expressions: 1, regulatory enzyme; 2, catalytic subunit; 3, regulatory (receptor) subunit; 4, effector and its enzymes

Fig. 5.9

Glycolysis and Krebs cycle divided into stages, with focus on their regulation and mutual coordination. Coordinating signals, line 1; regulatory signals, line 2; flow of products and substrates, line 3

5.3 The Characteristics of Process Coordination in Individual Cells and Organisms

Intracellular coordinating signals may be treated as counterparts of hormonal signals: they both share a similar mechanism of action, acting on receptors and altering their most fundamental property, i.e., sensitivity. However, the specificity of intracellular coordination pathways is somewhat different than in the case of hormones: such pathways rely on noncovalent interaction between signals and receptors (contrary to hormones, which usually trigger phosphorylation or other covalent modifications). It can therefore be said that intracellular coordinating signals exert influence on independent processes, but do not directly command them.

Another difference between both types of signals is the principle of unamplified action. Inside a cell, the distance traveled by a coordinating signal is usually short (negligible dilution of signal), negating the need for amplification. This issue is also related to the lack of a need for encoding. Encoding systems become necessary when the coordinating signal must undergo significant amplification—a process which also happens to aggravate any errors or distortions. The cell signals which effect intracellular coordination processes (products of the regulatory loops) are commonly called allosteric effectors.

Although the presented principles seem very generic, biological systems sometimes depart from them. Such departures involve both intracellular and hormonal signals (i.e., signals which the organism uses to control the function of individual cells). They are usually observed in circumstances where following established principles would result in unacceptable risk to proper transduction of the signal or the action of any component of the feedback loop (effector or receptor).

As a general rule, hormonal signals override intracellular signals. Hormones act by forming permanent, covalent bonds with proteins mediating signal and should therefore be treated as commands. However, in some cases obeying such commands involves the risk that the cell will become exhausted and unable to maintain its own vital parameters at an acceptable level. This issue is particularly important for enzymes which interfere with key cellular machinery. One example is the action of glucagon and insulin which control a critically important process (glycolysis) and can therefore easily drive the cell into metabolic distress. Both hormones strongly affect glycolysis by way of phosphofructokinase phosphorylation; however instead of directly interacting with phosphofructokinase-1, they act upon phosphofructokinase-2—an enzyme whose product forms a noncovalent bond with phosphofructokinase-1. Owing to this mechanism, a critically stressed hepatocyte may refuse to obey a command which threatens its own survival. This phenomenon is, however, something of an exception: similar safeguards do not exist in many other less important for cell survival processes, for instance, glycogen degradation, where the enzyme acting directly on glycogen (glycogen phosphorylase) is subject to covalent modification by hormones.

Atypical properties can also be observed in certain intracellular pathways such as the urea cycle, which involves signal coding. As noted above, intracellular signals are not usually encoded because they do not require amplification and are therefore largely protected against errors. Encoding may, however, become necessary whenever there is a significant risk of signal misinterpretation. Such a situation is observed in the urea synthesis process where an intense influx of nitric substrates into the hepatic cell may be uncorrelated with mitochondrial synthesis of carbamoyl phosphate.

As amino acid components of the urea cycle may be found in the mitochondrion regardless of this metabolic pathway, there is a risk that the coordinating signal could be misinterpreted if the messenger molecule were a simple amino acid. Hence, the urea cycle coordinator must be based on a substance which does not normally occur in mitochondria. This requirement is fulfilled by N-acetylglutamate which indicates the concentration of arginine in the cytoplasm. Synthesis of N-acetylglutamate is triggered by arginine, whose high concentration means that ornithine (a competitive arginase inhibitor) is not being transformed into citrulline at a rapid enough rate and that the process needs to be upregulated (Fig. 5.10).

Fig. 5.10

Coordination (dashed line) of mitochondrial and cytoplasmic stages of the urea cycle

We can therefore conclude that while standard signal transduction principles apply in most circumstances, special cases may require deviations from generic solutions.

5.4 Mutual Relation Between Cells and the Organism: Activation and Inhibition of Enzymes (Rapid Effects)

Interrelationship enables biological systems to perform advanced tasks; however it requires efficient cooperation and coordination.

The blueprints for mutual support determine strategies applied by individual cells and the whole organism in pursuit of their shared goal, i.e., homeostasis.

Cells express their specialized functions in response to commands issued by the organism. They are, however, autonomous, and if commands do not arrive, they may become independent, which is evidenced by changes in their behavioral strategies.

The mechanisms of cellular cooperation and coordination, along with their impact on energy transfer and storage processes, have been extensively studied. They can be easily traced, e.g., by observing the hormonal regulation of hepatic functions following consumption of food (fed state), during fasting and in intermediate phases. Given an abundance of nutrients (i.e., immediately following a meal), the organism releases a hormone (insulin) which forces hepatocytes to intensify their involvement in nutrient sequestration. On the other hand, if the organism is starving, liver cells are mobilized to inject additional nutrients into the bloodstream by consuming energy stores.

Regulation and coordination mechanisms involved in day-to-day functioning of the organism must be able to react to stimuli in a timely fashion. Thus, energy transfer and storage should be managed by activation and inhibition of existing enzymes rather than by changing their concentrations via transcription and translation.

Figure 5.11 presents the effect of insulin on liver cells—promoting sequestration of nutrients following a meal. In this case, the hepatocyte synthesizes fatty acids by activating carboxylase, acetyl-CoA, and ATP citrate lyases. Sequestration of triglycerides in the form of adipose tissue is enabled by insulin-mediated absorption of glucose into adipocytes. The combined effect of high blood glucose levels and the topping-off of glycogen stores effectively prevent the organism from further synthesis of sugars (gluconeogenesis).

Fig. 5.11

Control of sequestration processes in a liver cell. Dashed lines indicate regulatory and coordinating signals. The top right-hand inset depicts fluctuations in blood glucose levels as a result of the presented activity. Upregulated metabolic pathways are shown in color

Large-scale synthesis of fatty acids in liver cells requires significant quantities of ATP and involves oxidation processes where some of the nutrients are burned in order to power ongoing reactions. Thus, fatty acid synthesis is coupled to synthesis of ATP and NADPH. In fact, according to regulation principles, it is the overabundance of ATP and NADH which inhibits citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The role of this mechanism is to supply sufficient energy for sequestration processes. Most of the available glucose is converted into fatty acids, while the rest is consumed in the pentose shunt associated with production of NADPH.

During periods of starvation (Fig. 5.12) the role of glucagon increases (i.e., the insulin/glucagon ratio drops). The increased release of glucagon is a result of decreased blood glucose levels (note that maintaining steady concentration of glucose in blood is of critical importance to the organism). Glucagon works by reorienting the organism’s energy transfer and storage processes. Fatty acid synthesis is halted via inhibition of acetyl-CoA carboxylase and pyruvate kinase as well as activation of fructose-1,6-bisphosphatase. Blood glucose deficiency is compensated for by stimulation of gluconeogenesis, i.e., synthesis of glucose from amino acids, lactate, and glycerol derived from lipolysis. When oxaloacetate becomes scarce, the liver cell may draw additional energy from β-oxidation, ejecting ketone bodies (a byproduct of acetyl-CoA) into the bloodstream.

Fig. 5.12

Changes in hepatic metabolism resulting from reduction in blood glucose levels. The top right-hand inset depicts the fluctuating blood glucose levels. Dashed lines indicate intracellular regulatory and coordinating signals. Upregulated metabolic pathways are shown in color

During intermediate stages, i.e., when the blood glucose concentration is close to its biological optimum (approximately 5 mmol/l) and when insulin and glucagon are in relative equilibrium, the cell becomes independent. Its own regulatory mechanisms (based on noncovalent bonds) assume control, while the energy conversion processes occurring inside the cell are subject to allosteric regulation, primarily by ATP and NADH. The cell now focuses on maintaining optimal levels of ATP and NADH in its own cytoplasm and does not actively participate in ensuring homeostasis of the organism as a whole (Fig. 5.13).

Fig. 5.13

Regulation and coordination of energy conversion processes in a hepatic cell given a lack of coercive hormonal signals. The top right-hand inset depicts the fluctuating blood glucose levels. Dashed lines indicate intracellular regulatory and coordinating signals

An independent hepatocyte may synthesize fatty acids and glucose even when no hormonal signals are present; however in such cases, sequestration of nutrients is not the primary goal of the cell. Rather, the hepatocyte concerns itself with maintaining a steady concentration of energy carriers, treating synthesis as a convenient way to purge excess carriers. This change in strategy becomes evident in the readjustment of regulatory mechanisms involved, e.g., in glycolysis. Given an abundance of nutrients, glycolysis is activated by an external mediator (insulin) as a prerequisite of intensified sequestration. In an independent cell, the same process is controlled by citrate, which promotes fatty acid synthesis but—unlike insulin—also inhibits glycolysis. This means that a cell which is already rich in ATP may actively protect itself from further absorption of nutrients. Consequently, both ATP degradation pathways (fatty acid synthesis activated by citrate acid as well as glucose synthesis mediated by ATP and acetyl-CoA) remain open. The relatively low demand for glucose (compared with post-meal conditions) is satisfied by absorbing glucose directly from the bloodstream or by extracting it from glycogen with the use of glycogen phosphorylase, whose activity remains low but sufficient to cover the energy requirements of the liver cell.

Full independence of the hepatocyte is a short-lived condition and does not directly correspond to periods between meals (which can indeed be quite long). Cells can remain independent only when the contradictory activity of insulin and glucagon remains in equilibrium; however both hormones are active even when no food is being consumed—for instance, when strenuous physical exertion causes a significant drop in blood glucose levels.

Figures 5.14, 5.15 and 5.16 present organ collaboration in the scope of energy management, as reflected by the relation between glucagon and insulin levels. Hormone concentrations range from 0.5 μU pg⁻¹ (U, unit) following a meal to 0.05 μU pg⁻¹ during periods of starvation. During intermediate stages, when hepatocytes act independently, these values are close to 0.15 μU pg⁻¹.

Fig. 5.14

Collaborative energy management given an abundance of nutrients

Fig. 5.15

Collaborative energy management under conditions of low nutrient availability (decreased blood glucose level)

Fig. 5.16

Collaborative energy management in hepatocytes and in blood when the activity of insulin and glucagon is moderate

Tight collaboration between hepatocytes and the organism, regardless of the availability of nutrients (i.e., when nutrients are abundant, during periods of starvation and in intermediate stages), suggests that—according to the principles outlined above—cells and organism share similar goals and collaborate even though each of these units is subject to its own regulatory mechanisms. In both cases regulation occurs automatically, according to biological programming. Both types of regulatory loops act to stabilize the concentrations of vital substances—either in blood (the organism) or in the cytoplasm (individual cells) and mitochondria. Both systems (the organism and the cell) use specialized messenger molecules: the organism relies on hormones (for instance, insulin and glucagon), while the cell produces allosteric effectors (ATP, NADH, acetyl-CoA, malonyl-CoA, citrate, ADP, AMP, and NAD). Both systems also exploit specialized structural solutions which assist in regulation.

5.5 Mutual Support Between Cells and the Organism: Interdependence Related to Gene Expression (Slow Effects)

If both the organism and its environment are in a stable state, the concentrations of enzymes and other proteins involved in biological functions are defined by biological programming. Under such conditions, interrelationship may be realized by means of standard activation or inhibition of messengers (depending on their exchange dynamics).

If, however, the environment exhibits high variability and adaptations need to be introduced, quantitative changes associated with synthesis of additional substances become important. One example is hypoxia caused by severe blood loss, where synthesis of new blood cells is greatly intensified. Although erythropoiesis is a continually occurring process, it can be significantly upregulated by the action of certain proteins triggered by hypoxic conditions. This process involves differentiation of the erythrocyte line as well as actions related to hemoglobin synthesis (Fig. 5.17). Correct synthesis of hemoglobin, which consists of a protein unit and a porphyrin ring, depends on subprocesses which synthesize each of these components separately, as well as on the availability of iron. All subprocesses are subject to their own automatic control mechanisms, but they also exhibit cooperative behavior (Figs. 5.18 and 5.19). Since each occurs in a separate cellular compartment, they must all be coordinated in order to provide matching concentrations of various substances.

Fig. 5.17

Regulation of the biological response to hypoxia. Cooperation and coordination of processes occurring in an erythroblast. Solid line indicates cooperation. Dashed line indicates coordination. Raf-1, kinase; NF-E2, transcription factor

Fig. 5.18

Cooperation and coordination in the synthesis of heme and globin. Right-hand side, globin synthesis control; HCI, heme-controlled inhibitor; eIF, eukaryotic initiation factor. Inactive forms grayed out

Fig. 5.19

Transformation of glycine in a cooperative link with mitochondrial (heme) and external (purine) synthesis

Excess heme halts the synthesis process while downregulating the globin synthesis inhibitor. This enables continued synthesis of hemoglobin, consuming the available heme.

In a separate process, the hemoglobin synthesis apparatus (mostly based on ribosomes) also undergoes rapid expansion.

Hormonal signals induce transcription of mRNA and rRNA in the nucleolus. Synthesis of ribosomal proteins occurs in the cytosol. Once synthesized, proteins migrate to the nucleolus where ribosomal subunits are formed and released back into the cytosol. At the same time, mRNA strands travel from the nucleus to the cytosol, where the required proteins can be synthesized (Fig. 5.17). The pathway for signals which trigger hemoglobin synthesis involves transcription and is controlled by hormones.

It should, however, be noted that translation is subject to its own hormonal control, which also assists in the synthesis of hemoglobin. A hemoglobin-synthesizing cell includes a special mechanism capable of inhibiting translation. Its action is dependent on the concentration of heme: deficiency of heme halts further synthesis of globin and therefore acts as a coordinator (Fig. 5.18).

Heme synthesis (occurring in the mitochondrion) is also controlled by hormones as it depends on the availability of iron, which has to be drawn from outside the cell. Hemoglobin synthesis may proceed only if ample iron is available—thus, the response to hypoxia is conditioned by many independent (though mutually supportive) control processes.

5.5.1 The Structural Underpinnings of Interrelationship

The flow of signals which coordinate biological processes can be modeled on the basis of regulation principles.

As a rule, the path of each coordinating signal is a branch of some regulatory loop. Once released, the signal seeks out the receptor of another loop and adjusts its sensitivity to its own product. Inside the cell, signals are usually represented by products of the cell’s own controlled biological processes. On the other hand, organisms tend to rely on hormonal and neuronal signals, generated by specialized cells whose primary role is to assist in coordination (Fig. 5.20).

Fig. 5.20

Hormonal coordination of a complex response to a stimulus (such as stress). I, II, and III, hormonal cascade system levels; A, B, and C, effector loop cells

The need for correlated action becomes clear if we refer to the rules of regulation, determining the nature of both substrate and product of a given process (controlled by a feedback loop) and tracing the path of the product with respect to its biological purpose.

Coordination should be interpreted as a means of improving interrelationship whenever major deviations in the controlled quantity could be avoided (for instance, if a controlled substance exhibits undesirable activity or is otherwise toxic). However, coordination places an additional burden on biological systems as it forces them to provide receptors for external signals (Fig. 5.21). A solution to this dilemma comes in the form of receptor proteins, capable of binding their own product in addition to the products of regulatory processes to which a given system is coupled. Such proteins are almost exclusively allosteric, consisting of subunits and able to bind effector molecules in specific sites—just like hemoglobin, which can bind BPG (2,3-bipohosphoglycerate) as well as protons by way of the Bohr effect.

Fig. 5.21

Model view of an internal cell receptor complex (enzymatic regulatory subunits) capable of binding its own product (rectangles) as well as the product of another coordinating system (black circles). (a) Conditions prior to interaction with the coordinating system (low and high product concentrations). (b) Conditions after binding the signal of the coordinating system (allosteric effector)

The altered sensitivity of the receptor subunit is depicted as a change in the concentration of the controlled product, which, in turn, shuts down the controlling enzyme.

It appears that the need for coordination does not apply to all processes. Specifically, coordination can be avoided in processes which are of secondary importance to the cell. It is also unnecessary for sparse products, where significant fluctuations are not harmful to the cell or the organism.

Selected examples of metabolic pathways which require coordination are depicted in Fig. 5.22. As mentioned above, these are branching processes; moreover, their individual branches are often highly spontaneous and therefore irreversible. The figure presents processes (and parts thereof) which involve transmembrane transport (performed in separate organelles), where efficient transduction of products or substrates becomes difficult given significant variations in their availability or particularly rapid reaction rates.

Fig. 5.22

Examples of metabolic processes which require coordination. (a and b) Branching processes. (c) Variable inflow of substrates. (d) Process consisting of sub-stages separated by a membrane

Coordination of processes becomes easier if these processes share a common stage. The enzyme which catalyzes this stage (i.e., corresponds to a fork in the metabolic pathway) is naturally important for both intertwined processes. Such stages and the enzymes which catalyze them are called key stages and key enzymes, particularly if they affect vitally important processes. Examples include energy transfer mechanisms which involve synthesis of acetyl-CoA, glutamine, etc. These stages are natural targets for coordinating mechanisms.

A similar rule applies to hormonal coordination. A single hormone can control many processes by attaching itself to receptors exposed by many different types of cells (Fig. 5.23). Intracellular signal branching, resulting in the activation of interdependent processes, is also an example of complex coordination (Fig. 5.24). This type of action is observed, e.g., in the control of sequestration processes effected by insulin (Fig. 5.25). Each hormone, particularly a nonspecific one, can control a large number of processes simultaneously.

Fig. 5.23

Selective action of hormones upon “addressee” cells via specific receptors

Fig. 5.24

The impact of hormonal coordination on the cell

Fig. 5.25

The examples of branched hormone (insulin) signaling path inside the cell

5.5.2 The Role of Common Metabolite in Complex Process Coordination

If a coordination pathway simultaneously affects many linked processes, one of its components may become an indicator for a whole set of biological mechanisms which together generate the required effect. This strategic selection of a common marker is an important “technical solution” in many integration processes. As an example, let us consider the coordination of nutrient sequestration which depends on blood glucose levels.

Glucose is always present in bloodstream where its concentration can be readily measured. It is also an integrating component in the management of lipid deposits as a source of oxaloacetate required for degradation of acetyl-CoA. By penetrating adipose cells, glucose becomes a source of glycerol-3-phosphate, powering fatty acid sequestration (lipogenesis). As glucose processing is coupled to many energy management mechanisms, each such mechanism may affect blood glucose levels.

The storage of glucose in the form of glycogen is only meant as a buffer (no more than ~600 kcal is available at any given time, compared to the average basal metabolic rate of approximately 1600 kcal for a typical human). Thus, glucose sequestration and subsequent release must be a highly dynamic process. High concentrations of glucose affect the spontaneity of its intracellular metabolic pathways. Glucose is a good coordinator of energy management processes given its ubiquity, universality, and the critical role it plays in many tissues such as muscles, blood, and brain matter. It should also be noted that glucose is effectively the only nutrient whose concentration in blood is never subject to major fluctuations—thus, any changes in its levels are indicative of the state of energy management processes in the organism as a whole.

Glucose directly affects the release of two basic hormones: insulin and glucagon. Other hormones such as catecholamines or adrenal cortex hormones are less important for managing the organism’s energy stores. They usually come into play under stress conditions (catecholamines) or during prolonged periods of starvation when glycogen stores are depleted and the organism must draw energy from amino acids (adrenal cortex hormones).

Nutrient sequestration can be affected—via increased insulin release—by certain gastrointestinal hormones (incretins) such as GIP (gastric inhibitory polypeptide), whose production is stimulated by the presence of glucose and fatty acids in the small intestine: an interesting example of how the organism predicts and prepares itself for assimilation of nutrients.

The presence of substances which act as universal control indicators is biologically advantageous and can be observed in many different mechanisms. For instance, the entire nitrogen cycle in the E. coli bacteria is based on measuring the concentration of glycine and alanine. Bacterial glutamine synthetase consists of two subunits and has been shown to contain no less than nine allosteric binding sites upon which coordinating signals may act. These signals are, in turn, generated by cooperating processes and include carbamoyl phosphate, glucosamine-6-phosphate, AMP, CTP, and several amino acids (tryptophan, histidine, serine, glycine, and alanine). It should be noted that not all amino acids are actively involved in controlling the activity of the synthetase complex: from among the available molecules, alanine and glycine have been selected as the most common and the most intimately tied to the bacterial nitrogen cycle. A similar function appears to be performed by cyclins, whose synthesis is coupled (both in terms of reactions and substrates) to cell division, making them a good indicator of the division process.

5.5.3 Signal Effectiveness and the Structuring of Mutual Relations in Metabolism

In order to better understand the efficiency of regulation, cooperation, and coordination, we should consider the effectiveness of biological signals. This property is determined by the intensity of the signal and the sensitivity of its receptor; however it may also relate to the signal’s influence (the scope of activation or inhibition effects it produces).

Comparing the relative effects of various allosteric effectors on coordinated processes enables us to define a clear structure of coordination. The gradation of signal effectiveness is subject to certain rules. Hormones are usually far more potent than concentration-mediated signals (which characterize intracellular coordination) as their action involves signal amplification and covalent modifications of receptors. It is, however, more difficult to ascertain the relative hierarchy of the cell’s own signaling pathways. This hierarchy depends on the sensitivity of the regulatory enzyme to a given signal (which is constant) and on the concentration of signal molecules (which may vary).

For instance, we should expect that the effect of ATP—an allosteric effector involved in the cell’s energy management processes—upon mitochondrial enzymes will be stronger than its corresponding influence on cytosolic enzymes as ATP is highly diluted in cytosol compared to its mitochondrial concentration.

Glycolysis and the Krebs cycle involve several checkpoints where they may be inhibited by ATP. Proper description of these coordination processes requires a comparative assessment of ATP-mediated inhibition at each checkpoint for a given concentration of ATP.

In order to ascertain the specificity of coordination, we must first determine the affinity of the enzyme regulatory subunit (or other receptor systems) to the allosteric effector as well as the sensitivity of the entire system to the coordinating signal.

Variable signal affinity can be observed in many circumstances. One example is the coordination of ribosomal protein and rRNA synthesis, based on the ability of proteins to form complexes with rRNA and mRNA with differing levels of affinity.

Once synthesized, ribosomal proteins preferentially bind to rRNA (whose synthesis is mediated by hormones), creating ribosomes. This process continues until all available rRNA is consumed and free ribosomal proteins begin to build up. At this point freshly synthesized proteins start binding to the promoter fragment of their own RNA template, effectively halting the synthesis process (Fig. 5.26).

Fig. 5.26

Coordination of the synthesis of ribosomal proteins in the ribosome construction process. Ribosomal proteins exhibit higher affinity to rRNA and lower affinity to their own mRNA. Once rRNA is depleted, they begin binding to mRNA, halting their own synthesis

A similar problem occurs in coordination of cell division processes, where specialized proteins called cyclins are synthesized in tandem with division mediators. The energy and material expenses incurred by cyclin synthesis hamper the synthesis of proteins involved in cell division; however the latter group must be prioritized. Thus, cyclins amass at a slower rate, reaching their peak concentration only after the synthesis of the required division proteins and nucleic acids has concluded. Rapid degradation of cyclins triggers the next step in the division cascade. In this way a time- and energy-consuming process may act as a controller for other mechanisms associated with cell division. Instead of a “biological clock,” we are dealing with coordinating action, dependent—much like the cell itself—on the available energy stores and supplies of substrates.

5.5.4 Interrelationship in Times of Crisis: Safety Valves

Automatic regulation and coordination mechanisms allow cells to maintain proper substance concentrations and activity levels in spite of environmental changes. However, adaptability has its limits. A particularly acute crisis emerges when the cellular metabolic pathways are blocked by insufficient availability of oxygen. Energy requirements cannot be met if the cell is unable to metabolize nutrients. This is evidenced, e.g., in skeletal muscle cells, which on the one hand require an extensive network of blood vessels but on the other hand must be able to contract, limiting their ability to absorb oxygen from the bloodstream. One consequence of this apparent paradox is the synthesis of lactic acid. Given an insufficient supply of oxygen, this byproduct cannot be further metabolized and therefore builds up in the muscle cell. High levels of lactic acid threaten the cell by lowering its pH, increasing osmotic pressure, and—most importantly—inhibiting glycolysis by altering its oxidation-reduction potential and interfering with enzymes which participate in breaking down glucose.

If the cellular regulatory mechanisms are unable to prevent excessive buildup of lactic acid, the only solution is to expel this unwanted byproduct into the bloodstream where acidification can be counteracted by other regulatory systems (such as the liver, capable of neutralizing lactic acid). This mechanism can be compared to a safety valve which prevents the destruction of an overpressurized boiler by venting excess steam. A similar situation occurs during starvation when the relative deficiency of oxaloacetic acid inhibits the Krebs cycle and hampers further metabolism of acetyl-CoA (extracted from fatty acid degradation), resulting in increased production of ketone bodies which must also be ejected into the bloodstream. Oxaloacetate acid synthesis is largely dependent on the supply of glucose (a key source of pyruvate).

Many organisms have evolved coordination mechanisms which enable them to handle the byproducts of metabolism appearing in the bloodstream. Naturally, single-cell organisms have no such problems and simply expel unneeded substances (alcohol, lactate, acetoacetate, etc.) until their concentration in the environment halts cell proliferation.

Lactic acid is produced mainly by muscle cells and erythrocytes and metabolized by hepatocytes. A similar mechanism applies to ketone bodies, which are generated in the liver and broken down in the brain, heart, kidneys, and skeletal muscles.

5.6 Specialization of Cell Interrelationship

Specialization results in functional differentiation and requires active relationship if the differentiated tissues are to constitute a biological entity (a cell or an organism). Examples of specialization include organelles (compartments) in the cell and organs in the organism, although each of these groups follows different principles. Cell organelles are self-contained intracellular subunits which assist the cell in achieving its strategic goals. They facilitate cooperation and coordination but also enable separation of processes which would otherwise interfere with one another (such as synthesis and degradation). An example of this mechanism is β-oxidation and synthesis of fatty acids. Furthermore, some processes are potentially harmful to their environment and need to be isolated (this includes lysosome activity).

Given our present state of knowledge, it would be difficult to accurately and unambiguously describe the strategies of intracellular compartmentalization. Some enzymes change their location as a result of evolutionary processes. For instance, rhodanese has migrated from the cytoplasm to the mitochondrion in the course of evolution. Explaining this shift would require good knowledge of the interactions between rhodanese and the intracellular environment; however it also proves that at least in some cases enzyme location is not crucially important.

In light of the above facts, we may ask why glycolysis—a degradation process—is not itself restricted to the mitochondrion. The answer lies in the natural strategies applied to separation and aggregation of intracellular processes. Assuming that separation of contradictory processes, while useful, is not of key importance enables us to consider other factors which may influence their location. In the presented case, synthesis of ATP and other energy carriers is more important than any potential benefits derived from physical separation of the relevant metabolic pathways (note that mitochondrion-independent glycolysis is an important source of ATP).

A crucial property of glycolytic ATP synthesis is its independence of an external oxygen supply. In contrast, the Krebs cycle and conjugated with it other mitochondrial processes slow down whenever oxygen becomes scarce. Under such circumstances an oxygen-free energy supply becomes critically important and cannot be subordinated to mitochondrial processes. Of course, relinquishing physical separation of glycolysis components introduces the need for accurate control and coordination, which in turn explains the importance of regulatory processes associated with glucose metabolism.

Considering the organism as a whole, specialization should be understood as a consequence of cell differentiation. On this level interrelation means the difference between a pool of random cells and an efficient organ which can assist its host organism in maintaining homeostasis. In animals cooperation is enabled by transporting substances via the bloodstream, while the coordination of differentiated tissues occurs by way of hormones or (in certain cases) neuronal signaling.

The function of the organism is based on a homeostasis program “encoded” in the structure of its receptors.

In general, the term organism is applied to any group of independent, differentiated units which together constitute a higher-level structure and coordinate their actions in pursuit of a common goal. This process opens up avenues of development not available to undifferentiated units such as cells.

The word organism may also be used in relation to the social structure of a state or an insect colony comprising individual castes which communicate through chemical means (pheromones) and through physical contact. Examples of this phenomenon include ant and bee colonies.

In humans, the “colony” model can best be observed in the immune system, which also consists of specialized “castes” of cells—workers (B and T lymphocytes) and soldiers (monocytes, killer cells, and phages). Both groups originate from similar nonspecific proliferation centers (hematopoietic stem cells, which can be compared to ant colony queens), and both exhibit the ability to gain a specific “personality” via interaction with the histocompatibility complex (much like pheromones, lymphokines, or direct physical contact among social insects). Moreover, immune system cells are also able to acquire experience.

The level of similarities between such disparate biological systems suggests a common organizational strategy based on the principles of information theory.

5.7 Microbiome

A peculiar mode of co-action is encountered in human and animal organisms which form mutualistic relationships with bacteria present in their organs—especially in the gut. The number of bacteria present in a typical organism is typically far greater than the number of cells which make up the organism itself. Clearly, the presence of such a large quantity of microbiota must have an effect on the organism. Given that—under normal circumstances—these microbiota do not cause disease and in many cases actively promote the organism’s well-being, their activity should be regarded as beneficial, even if they are not integrally linked to the organism. This so-called microbiome is highly diverse, and its composition strongly depends on the host’s dietary habits, which is why it varies from organism to organism. The microbiome’s role is not limited to digestion. While it clearly influences metabolism, it is equally important as a source of stimuli for the immune system, exploiting the activity of B cells migrating from germinal centers. As a result, this bacterial system enjoys a mutualistic relationship with its host.

5.8 Bacterial Bioengineering Techniques

Explaining the structure of the genetic code and the basic principles involved in its construction paved the way toward possible interventional procedures, roughly referred to as “genetic engineering.” The necessary prerequisite of such interventions is the ability to accurately cleave the DNA helix in specific places, in order to introduce the desired modifications and mutations, and also to copy specific fragments of the genetic material.

As can be expected, genetic engineering techniques reflect mechanisms which have long been employed by prokaryotes. Bacteria in particular have evolved intracellular defense mechanism against their principal foe: phages, i.e., entities which penetrate bacterial cells, injecting their own DNA. Such mechanisms work by identifying and destroying alien DNA.

Since bacteria cannot rely on external defensive structures (unlike higher organisms), they had to devise intracellular mechanisms capable of detection and selective hydrolysis of alien DNA. This mechanism has been initially repurposed in human genetic engineering. Its specificity is achieved by accurate action of bacterial enzymes known as restriction enzymes, capable of recognizing palindromic sequences. Such sequences are characterized by repetition and symmetry, as illustrated Fig. 5.27.

Fig. 5.27

Schematic view of using restriction enzymes to cleave DNA in specific places, leaving behind so-called sticky ends, as well as employing bacterial plasmids as carriers of external DNA, pasted in the process of replicating genetic material

Restriction enzymes are capable of recognizing specific sequences and work by cleaving DNA strands in two slightly offset places, leaving behind an uneven tip known as a sticky end. These ends are indeed “sticky” given that they may spontaneously, noncovalently reassociate, restoring the original connection, which is subsequently covalently reinforced by ligase (Fig. 5.28).

Fig. 5.28

Mechanism of DNA amplification with the aid of bacterial plasmids

Palindromic sequences are ubiquitous and can be found in DNA derived from various organisms. By cleaving its affine palindrome, the restriction enzyme fragments DNA. The resulting fragments may reassociate while incorporating other external DNA (created in a separate cleaving process, mediated by the same enzyme), since all its ends remain “sticky”—which is due to the fact that they share a complementary sequence.

This mechanism, discovered in the bacterial domain, has been applied to replicate genetic material and to synthesize specific proteins.

The bacterial genome is arranged in a large circular chromosome and many smaller, chromosome-like circular structures called plasmids, some of which are capable of replication. Genetic engineering utilizes entities known as vectors—small plasmids which have been modified to contain a single palindromic site corresponding to a specific restrictase. By “opening up” the circular plasmid, the restriction enzyme primes it for attachment of external DNA (cleaved by the same restrictase and therefore possessing matching sticky ends). The modified plasmid, containing alien genetic information, is then reintroduced into the bacterial cell and replicated. This “cuckoo’s egg” strategy enables us to produce a large quantity of specific genetic materials, which is inserted into the plasmid and subsequently isolated using the same restrictase as before (Fig. 5.28).

Another more advanced technique which bacteria employ to combat phages and which can also be utilized by genetic engineering is referred to as CRISPR (clustered regularly interspaced short palindromic repeats). It assumes the form of a genetically encoded matrix for a RNA sequence which directs the attached CAS nuclease to cleave both strands of the phage’s DNA at a specific point. The operon involved in this process represents a unique intracellular bacterial defensive measure, capable of remembering specific features of its attack targets, including the phage’s DNA. The associated information packets, encoded in the bacterial chromosome, carry information of alien (phage) DNA with which the bacteria have previously come in contact. Such information is interspersed with short palindromic sequences. The resulting RNA can recognize the phage’s DNA, since its own DNA matrix is, in fact, the phage’s DNA which has been fragmented, integrated with the bacterial genome (bracketed by palindromic fragments) and subject to inheritance as a form of long-term database for the bacterial anti-phage defense system. In its active form, the system assumes the form of RNA associated with CAS nuclease (in the case of the commonly analyzed Streptococcus pyogenes—CAS⁹), which becomes active by binding to RNA. CAS nucleases consist of two domains referred to as HNH and RuvC. Each cleaves a specific strand of the phage’s DNA, with the HNH domain targeting the strand directly referenced by the bacterial RNA (in complex with CRISPR).

Recognition of alien DNA is not, however, based solely on interaction of a specific RNA strand with the target DNA. An additional condition involves interaction between nuclease and a short (two to four nucleotides) fragment of the target DNA, referred to as the protospacer adjacent motif (PAM). PAM is directly proximate to the fragment which corresponds to the RNA template, and its interaction with nuclease initiates splitting of the target DNA strands, enabling RNA to form a complex with one of them.

The structure and activity of the CRISPRCAS system both confirm the ubiquitous mechanism whereby RNA is used as a “pointer,” leading active proteins to their intended targets, whether DNA- or RNA-based (Fig. 5.29).

Fig. 5.29

Linking together products of the CRISPR bacterial operon with HNH and RuvC domains of the CAS nuclease in an attack on a recognized DNA sequence

Eukaryotic cells employ a similar system, centered upon the so-called Argonaute protein, which exhibits nuclease activity. This system has recently attracted increased attention as a possible alternative for CRISPRCAS, endowed by greater specificity and smaller size, both of which facilitate integration with carrier viruses.

The CRISPRCAS system has proven highly versatile owing to its capacity for easy modification and the resulting wide range of applications in biology and medicine. Modifications may involve the RNA itself as well as the interacting proteins. Some authors have reported successful attachment (via fusion) of further proteins, which provide additional activity. CRISPR has so far found use as an extremely sensitive detector of viral infections, revealing the presence of viruses in the organism long before any clinical symptoms emerge. Nevertheless, its in vivo use for introducing targeted genomic modifications encounters obstacles related to false positives and off-target mutations. The technique is still being perfected, and its great promise suggests that CRISPR may, in the near future, become the mainstay of clinically relevant genetic engineering techniques.

In vivo techniques also employ AAVs (adeno-associated viruses) as carrier vectors.

Another powerful technique allowing amplification of DNA synthesis using selected DNA chain fragments as the template is known as polymerase chain reaction (PCR). It was devised in 1984 by Kary Mullis. The technique derived from thermophilic bacteria heat stable polymerase allowing polymerization of DNA after having its chains separated simply by temperature (95 °C, 5 s) applies. The rapidly lowered temperature to 54 °C allows then the attachment of primers, and when temperature is again elevated (72 °C, temperature optimal for polymerase activity), the process of DNA synthesis starts. This procedure may be repeated again and again producing the increasing amount of selected DNA fragments. Thus finally in the presence of substrates (active nucleotides), primers kept in the access, and heat stable polymerase, only the suitable manipulation of temperature is necessary to run the DNA synthesis.

5.9 Hypothesis

5.9.1 Carcinogenesis

The mechanism of neoplastic transformation remains unclear despite numerous efforts to understand it. In particular, an integrated approach to the problem from a molecular perspective is yet to be devised.

It seems clear that the process is driven by mutations; however, mutability is a very broad concept, and its links to neoplastic transformation are not understood in detail. In most cases a cell which has undergone such transformation is found to contain numerous mutations, causing significant damage to its genome. It would be natural to expect such cells to exhibit poor viability—which runs counter to the observation that cancer cells are often highly active and prolific. What is more, these active, rapidly dividing cells do not resemble ordinary building blocks of the organism—they do not conform to restrictions imposed by the organism and should instead be treated as alien, harmful agents.

Uncontrolled division and loss of intended biological function are the hallmarks of genetic reprogramming which causes otherwise ordinary cells to break free from the rules to which all healthy cells must conform. The organism is a system composed of varied but cooperating cells, all of which carry out a common program, ensuring homeostasis and enabling the organism to function as a coherent whole. This situation may be compared to a political system, involving a state and its citizens—distinct but collaborating entities. The latter are subordinate to the former, while the former ensures coordinated action.

In both cases—i.e., with regard to cells as well as citizens—subordination relies on signals which are mandatory in character. Thus, both systems have a hierarchical nature. In the case of the state, this hierarchy is enforced by its administration, whereas in an organism, it assumes the form of signals, which—unlike intracellular signals—depends on covalent bonding (independent of substrate concentration) and very strong amplification, typically facilitated by cascading processes.

Given such conditions, it becomes imperative to prevent the command signal from persisting beyond its intended timeframe. As a result, cells have evolved mechanisms which enable such signals to be attenuated or suppressed entirely.

This function falls to the so-called suppression systems associated with signaling pathways (Fig. 5.30). Their action usually manifests itself as a GTPase-dependent process—as in the case of protein G, for which GTP activates signals, while its hydrolysis attenuates them or, alternatively, the action of phosphatases which suppress phosphorylation-related activity and determine the activity of kinases, such as tyrosine kinase. This suppressive effect may be present at all times and become amplified along with the amplification of command signals. Examples of such systems include suppressors involved in cell proliferation, such as p53, APC, axin, and others (Fig. 5.31).

Fig. 5.30

Command signals guiding proliferation, with their associated suppression components (in rectangular frames)

Fig. 5.31

p53 and its basic cellular interactions, revealing the effect of mutations. The figure illustrates the permanent standby activity of the p53 suppressor system in the scope of cell proliferation

The role of suppressor systems is critical in ensuring balanced and controlled command signal activity. Mutations which inactivate suppressor genes in the cell division process lead to uncontrolled proliferation and are implicated in neoplastic transformation.

In the course of evolution, independent cells became involved in higher-order structures—organisms—and had to adapt their “programming” accordingly. In particular, cells which form part of an organism had to:

• 1. Undergo differentiation, subject to the principles of epigenetics.

• 2. Become susceptible to controlled senescence and apoptosis to enable selective development and supersedure of cells which have accumulated too many genetic defects during their lifetime. This goal is achieved by progressive downregulation of telomerase, which imposes an effective limit on the number of divisions a cell can undergo, or by mechanical elimination of cells from the organism—e.g., by exfoliation of epithelial cells,

• 3. Limit their proliferative activity in accordance with “stop” signals received from neighboring cells, in order to ensure formation of properly structured organs. This mechanism is also known as contact inhibition.

• 4. Optimize their respiratory efficiency.

The emergence of organisms could be regarded as a major evolutionary breakthrough—as well as a breakthrough in terms of cellular programming, where individual cells had to forgo some of their autonomy in order to obey commands issued by the organism.

Neoplastic cells break free from this command hierarchy while retaining their viability and ability to proliferate (despite being saddled with numerous mutations). This phenomenon could be compared to an act of rebellion against the organism. Such selective outcome of mutations may be explained by referring to the process of evolution and to the specific nature of certain mechanisms which emerged in its course.

While eukaryotic cells evolved approximately 1.6 billion years ago, organisms have a much shorter history, with the earliest such biota dating back to the Ediacaran period (ca. 650 million years ago). Complex organisms—initially of the marine variety and later land-based—appeared in the Cambrian period (ca. 100 million years ago). Thus, individual cells had a much longer time to fine-tune their internal regulatory systems and ensure their stability than organisms, which rely on broader regulatory mechanisms to ensure coordination among various types of cells (Fig. 5.32).

Fig. 5.32

Timeline of the evolution of organisms against the backdrop of geological periods

Sufficient stability may be conferred by an evolutionarily conditioned set of genes which do not mutate easily due to the presence of proteinic safeguards (nucleosomes) and DNA packing or efficient DNA repair mechanisms. The same effect may also be achieved by systems capable of eliminating cells which contain damaged DNA or are otherwise aberrant (apoptosis). However, a similar outcome is produced by suitable suppression of command signals, which—among others—prevent excessive proliferation and thus protect DNA from incurring unacceptable mutational pressure. Nature provides us with many mechanisms which may potentially mitigate neoplastic pathologies. Examples include anti-cancer compounds which influence regulatory mechanisms related to cell proliferation, such as thalidomide or rapamycin. Given the availability of such mechanisms, an interesting question is why neoplastic transformation occurs at all, and why it poses such a serious threat to organisms. The simplest answer is that there has not been enough time (in evolutionary terms) for organisms to evolve foolproof protective mechanisms. Another point which can be made is that it is often counterproductive (again, in terms of evolutionary gains) to prolong the lifespan of individual organisms—thus, no corresponding mechanisms have evolved over the long term.

DNA mutation is a progressive process, spurred by random errors which occur naturally during the replication cycle. A serious threat emerges when the rate of mutations is accelerated—whether by external or internal factors. The former include ionizing radiation, aggressive chemical compounds, or viral infections, while the latter mainly involve free radicals (a natural byproduct of the respiratory cycle) or hormonal deviations. DNA’s susceptibility to mutagens increases when immediate repair is hampered or impossible. This often occurs when a single DNA strand is exposed for a long time in the process of replication or transcription. A rapidly dividing cell is particularly prone to accumulating mutations—especially, when there is not enough time to properly pack its DNA and repair emerging errors. Damage to genes which participate in suppressing command signals in division control pathways is especially dangerous. Breakdown of proliferative control—which is the main mechanism by which the organism exerts control over individual cells—causes the cell to become independent of the organism and switch over to its evolutionarily conditioned survival program, with no regard for the needs of the organism as a whole. This phenomenon can manifest itself through (1) ignoring command signals, particularly contact inhibition effects; (2) activation of telomerase, which effectively renders the cell immortal; (3) regression of differentiation through remodeling of chromatin; and (4) preference for fermentation over aerobic respiration (the so-called Warburg effect).

Mutations which emerge in the course of replication preferentially affect those signaling pathways which are evolutionarily younger and devoid of suitable safeguards—such as inherent structural stability and resilience of attenuating components of signaling mechanisms, capable of counteracting excessive proliferation. Disabling such mechanisms renders the cell susceptible to uncontrolled divisions.

The need for and operation of signal attenuation mechanisms may be schematically demonstrated by referring to the emergence of statehood in societies and to its subsequent democratization. Here, analogues of attenuation mechanisms may be found in institutions designed to counteract uncontrolled exercise of executive power, such as the Constitutional Tribunal, the Ombudsman’s Office, the National Judiciary Council, some media outlets, etc. Creation and fine-tuning of systems which act in opposition to top-down command pathways and can attenuate them require a long time.

Mutational changes in signaling pathways are more commonly observed in cases of cancer than pure statistics (e.g., Gaussian distribution) would suggest. One example is the Tp53 gene, found to be damaged in over 50% of cancers. Such nonrandom susceptibility to damage in mechanisms which control command and suppression activity is also evident in the irreversibility of neoplastic transformation. Proof of selective susceptibility to mutational pressure is found in the different rates at which tumors are found in various types of organisms and individual organs. Here, edge cases include laboratory mice (which are highly susceptible) and African mole rats, which can live over 30 years and almost never develop tumors. In the latter case, high resilience is thought to result from particularly efficient protein synthesis and folding processes, believed to be assisted by RNA S28-mediated ribosome activity and synthesis of macromolecular hyaluronic acid, associated with upregulation of p16 and p27 genes, which improve the efficiency of contact inhibition. This confirms that mechanisms reducing the risk of neoplastic transformation may emerge in the course of evolution. As organisms age, the risk of carcinogenesis increases due to accumulation of mutations but also—likely—due to reduced efficiency of protein synthesis and degradation mechanisms, all of which are vitally important.

Thus, neoplastic transformation can be described as a process through which cells break free from their link with the organism and revert to existing as individual, independent entities while also forfeiting their differentiation (partly or fully). According to this hypothesis, tumor cells “reenact” an evolutionarily ancient program which existed prior to the emergence of complex organisms.

From the point of view of pathophysiology, the direct cause of neoplastic transformation is usually associated with excessive, unchecked cell divisions, increasing disorder in the arrangement of chromatin and the resulting increases in mutation rate. Interestingly, the general characteristic of transformed cells tends to be similar despite differences in their initial epigenetic configuration. When comparing genes which undergo mutations in various types of cells, leading to their neoplastic transformation, it turns out that their functional properties are also similar. In short, these genes can be divided into three groups: (1) genes associated with signaling pathways (mainly suppressors); (2) genes associated with maintaining proper DNA structure and DNA repair, synthesis, and packing; and (3) genes directly associated with epigenetic processes and chromatin structure (Fig. 5.33).

Fig. 5.33

Groups of genes with different origin but similar functional profiles, all participating in enforcing cell-organism integration and often implicated in neoplastic transformation (as mutants): I, ureters; II, large intestine; III, leukemia

Mutations in this set of genes often result in accelerated cell division since they mainly affect signal suppressors. Increased mutability is a natural consequence, and in this process the evolutionarily younger genetic programming—which is not equipped with strong protective mechanisms—is often disrupted, leaving more ancient processes intact.

Maintaining homeostasis in the scope of signaling pathways calls for a set of interlinked suppression systems. The resulting network is quite complex and appears more susceptible to disruption than the command system itself. Notably, certain gene families are strongly implicated in mutation-induced carcinogenesis—these include Tp53, WHL, FLT3, APC, ARIDIA, PIK3CA, NPM1, KRAS, FBXW7, MLL, and KDM6A. Tp53 is particularly noteworthy as it is closely tied to apoptosis and to suppression of telomerase. It therefore seems likely that the main function of genes which often undergo mutations in cases of cancer is to safeguard the link between the individual cell and the organism to which that cell belongs. Inactivation of such genes opens the door to neoplastic transformation.

5.9.2 The Criteria of Life

Expanding biological knowledge allows us to study the strategies used by nature. However, we still lack a satisfactory definition of the very notion of life. Self-organized, autonomous biological systems are usually said to be “alive”; however this inclusive criterion does not posit any specific boundaries. The question whether certain structures (particularly subcellular ones) are alive remains unresolved. Moreover, there is still no scientific consensus regarding the procedural definition of life.

As can be expected, this issue has been approached by many great researchers over the course of history. It seems that the definition which most closely matches our modern scientific knowledge is the one proposed by Claude Bernard, who focused on the independence of biological systems, i.e., their ability to function under varying environmental conditions. Increasing freedom of action—corresponding to increases in the complexity of biological systems—appears to be the most general defining characteristic of life. Freedom of action can therefore be treated as a measure of the complexity of an automaton, reflected by its capability to make autonomous decisions.

The properties and characteristics of structures which we call “alive” may be explained by their automatic behavior. It would, however, be misleading to fully equate a biological system to an automaton. Modern technology, particularly robotics, creates automata which sometimes closely resemble living organisms, blurring the border between life and technology. Nevertheless, biological entities differ from even the most intricate robots: they are governed by specific rules which determine the structural and functional differences between various types of organisms. Life is subject to certain restrictions not observed in robotics. For instance, a fundamental aspect of nature is the phenomenon of programmed death—the result of biological strategies which focus on the survival of the entire systems rather than individual entities, as a means of ensuring harmony and maintaining equilibrium. All properly constructed biological entities must include death as part of their natural programming. In cells, this programming is evidenced by limitations in the possible number of divisions, along with senescence and apoptosis. The inclusion of a terminating function (death) in a biological program calls for a clock-like mechanism, capable of measuring time. Cyclical processes, such as the circadian rhythm, which fulfills the role of a “biological clock,” are crucial for living organisms. Entities whose programming does not involve the possibility of death—for instance, tumor cells—are considered pathological. (Note, however, that this group does not include gametes, where only the genetic information can be called “immortal”—individual gametes can and do undergo controlled death.) Cells which do not age or die are inconsistent with the principles of nature. In this aspect, the definition of life must necessarily be enumerative: nature itself determines what is and what isn’t alive. Close study of biological entities straddling the border between the animate and inanimate worlds reveals the importance of controlled death in natural systems.

By analyzing and comparing the genomes of primitive organisms and other biological entities, we can quickly derive a minimal set of genes and functions required to support what is commonly understood as life. Let us focus on the erythrocyte, which is clearly a biological structure (even though it cannot reproduce). The fact that it derives from a fully functional cell makes it a good test subject. Erythrocytes retain automatic control of metabolic processes (steady state), maintaining proper ion gradients and oxidation-reduction potentials which enable them to perform their function. They also actively counteract deviations which emerge as a result of cyclical migrations from the lungs to other tissues. What is more, they are subject to rapid degradation in a process which clearly defines the end of their usefulness (Fig. 5.34). Maintaining a steady internal environment despite drastic external changes is an important characteristic of healthy erythrocytes. In a human organism, each erythrocyte visits the lung capillaries approximately once per minute, releasing carbon dioxide and protons (this is called the Haldane effect). The oxygen-rich environment of the lung increases the odds of encountering dangerous reactive compounds (oxidative stress). By the same token, traveling through the acidic environment of metabolically active tissues alters the production and degradation rates of various intermediate substances in key metabolic pathways. Of note is also the relative difference in temperatures—from 28 °C in lung tissue to 40 °C in certain areas of the liver. In the kidneys, erythrocytes are subject to a rapid increase in osmolarity. Finally, the width of a typical erythrocyte is larger than the diameter of a capillary which means that traveling through narrow capillaries causes friction and distorts the cellular cytoskeleton. Counteracting all these changes requires the cell to expend energy (in the form of ATP and its derivatives reduced by NAD and NADP).

Fig. 5.34

Average duration of life of erythrocytes in various animal species

ATP synthesis in human erythrocytes depends on glycolysis (Fig. 5.35). As erythrocytes lack mitochondria, this process also produces excess lactic acid. Regulation of glycolysis in erythrocytes is particularly susceptible to changes in pH. Increased acidity inhibits the glycolysis initiator enzyme (phosphofructokinase-1) as well as certain other allosteric enzymes: pyruvate kinase and hexokinase. Lower efficiency of pyruvate kinase, as compared to phosphofructokinase-1, causes more metabolic intermediates to flow through the oxidative component of the pentose-phosphate cycle. In addition, lower pH also reduces the intensity of the Rapaport-Leubering cycle by inhibiting biphosphoglycerate mutase. Together, these phenomena result in increased production of reducing equivalents, protecting the cell from internal oxidation. This effect is particularly important given the relative abundance of oxygen in erythrocytes traveling from the lungs to other tissues. It ensures that iron is maintained at +2 oxidation, prevents protein clumping, and reduces damage to biological membranes. It should be noted that old erythrocytes exhibit slightly lower internal pH compared to younger cells.

Fig. 5.35

Red cell metabolism standing in opposition to oxidative and osmotic stresses

The erythrocyte fulfills a rigorous biological program which does not provide for substantial freedom of action. It can be compared to a biological “robot” which does not necessarily have to be called alive. Nevertheless, the erythrocyte can also be said to possess some properties of life, most notably a “death trigger” (Fig. 5.35). The abrupt cessation of biological function proves that cell death is not simply a result of stacking internal damage, but instead results from a timer-like mechanism which leads to programmed suicide. The need for such a mechanism is dictated by the necessity of replacing senescent cells and controlling the degradation of cells following their death. In humans, approximately 200 billion erythrocytes die each day, releasing a substantial quantity of hemoglobin which could easily overwhelm the organism’s compensatory mechanisms. Controlling the degradation of erythrocytes is therefore very important. Much like apoptosis, the process is biologically programmed. In order to distinguish it from the death of eukaryotic cells, it has been called eryptosis. It occurs as a result of osmotic or oxidative stress or when the cell cannot produce sufficient energy to power its membrane pumps. Eryptosis is characterized by an increase in the concentration of calcium ions. Only approximately 0.06%–0.4% of erythrocytes suffer accidental (unprogrammed) death each day, regardless of age.

According to the presented criteria, the erythrocyte—unlike viruses—may be called a living entity because it performs its function automatically and is subject to programmed death.

Scientific consensus holds that in eukaryotic cells senescence and death are controlled by a mechanism which involves progressive shortening of telomeres. However, erythrocytes lack chromosomes and must instead rely on a different process. It is known that erythrocytes (including those stored in donor blood) progressively shed fragments of their membranes. This phenomenon is most probably associated with biologically conditioned instabilities in certain areas of the membrane. It becomes more rapid in an acidic environment and seems to correspond to increases in ambient temperature. Damage sustained by the membrane lowers its active area and causes loss of protein microvesicles. After a certain number of cycles spent carrying oxygen from the lungs to other parts of the organism, the erythrocyte is no longer able to perform the required metabolic actions during its brief stay in each of these tissues. The maximum number of lung tissue cycles is similar in most organisms.

The relation between the erythrocyte’s lifespan and the weight of the animal is also fairly well known and enables us to derive a mathematical expression for its longevity. Cyclical reductions in the cell’s surface area and volume eventually trigger changes which turn the erythrocyte into a target for phages. As the cell sheds fragments of its membrane, it also loses substances protecting it from being absorbed by the reticuloendothelial system in the liver and spleen. The lower the number of microvesicles in the membrane, the higher the concentration of certain integrated proteins, which eventually begin to clump. Such protein aggregates are recognized by immunoglobulins, resulting in the erythrocyte being removed from the bloodstream. Aggregation of integrated proteins also impacts the activity of enzymes participating in the cell’s energy management processes. An example is the AE1 protein, whose aggregates inhibit certain glycolytic enzymes in senescent red blood cells. The same protein also facilitates anion transduction—binding to enzymes renders it unable to maintain a proper ion gradient inside the cell. Decreases in surface area affect the overall shape of the cell and—consequently—the distribution of cytoskeletal proteins. A dying erythrocyte is characterized by increased levels of phosphatidylserine in the outer phospholipid membrane layer. This is a result of lowered availability of ATP, which is required by enzymes responsible for moving this phospholipid into the inner membrane layer (flippases). A sudden increase in the concentration of calcium ions, characteristic of eryptosis, activates an enzymatic protein called scramblase, which also contributes to the exposure of phosphatidylserine. Together both processes trigger mechanisms which degrade the aging cell and clear it from the bloodstream.

Inhibition of any component of interlinked metabolic pathways results in metabolic dysregulation which may lead to cell death. This type of process is observed in diseases which lower the expected lifespan of certain cells—hemolytic anemias caused by deficiencies or inactivation of glycolytic enzymes, defective pentose-phosphate cycles, as well as cytoskeletal abnormalities and dysfunction of integral proteins which constitute biological membranes. Figure 5.31 presents a simplified diagram of erythrocyte metabolic pathways.

The mechanism which triggers erythrocyte death is specific to this group of cells, proving the importance and universality of programmed death. It seems that protein microvesicles perform the function of a biological clock. Once triggered, the death process closely resembles apoptosis. Dead cells are removed from the bloodstream by phages. It seems that while automation imparts biological structures with certain properties of life, the true requirement of inclusion in the animate world is the presence of a programmed death mechanism.

We can therefore conclude that a natural entity is alive if it exhibits autonomy as a result of automatism and follows a programmed mechanism of action which includes a timed death trigger.

It is worth noting that living organisms make frequent use of clock-like mechanisms. Such oscillators are usually based on negative feedback loops, but they also involve positive feedback loops in order to ensure suitable signal properties. They exploit specific biological phenomena as a means of measuring time, often with far-reaching consequences. The ubiquitous nature of oscillators found in all types of cells (including bacteria) seems to indicate the reliance of nature on cyclical processes. The frequency of oscillations varies—from one cycle per second to one per day or even one per year. In the human organism, a particularly important task is performed by the circadian rhythm controlled by a so-called master clock, which coordinates the action of other systemic oscillators. Its development is closely linked to the evolution of life on Earth, governed by the day-night cycle. The circadian rhythm works by anticipating changes in activity associated with various parts of the day, which is why the master clock located in the suprachiasmatic nucleus (NCS) is linked to the retina through a dedicated neural pathway.

A different type of biological oscillator can be found inside cells. Intracellular oscillators measure various frequencies (driven, e.g., by changes in the activity of glycolytic enzymes, calcium ion levels, etc.) and exploit feedback loops which link information stored in DNA to protein products. The master circadian clock seems to be affected by cyclical changes in the activity of transcription factors (CLOCK and BMAL-1) which, in turn, induce the transcription of repressors (Per and Cry proteins); however the role of this mechanism is not precisely known. To date research suggests a connection between the action of oscillators and intracellular metabolic pathways. The universality of this phenomenon may indicate the fundamental importance of actively reinforcing certain biological processes, which—being subject to automatic control—exhibit a natural tendency to slow down having reached a preprogrammed level of activity.

Further research may bring an answer to the question whether oscillators are an indispensable component of living organisms and whether their presence can be included among the criteria of life.