[Necessary sites: identical duplication of living organisms]. / Il caso e la necessità: la duplice identità degli organismi viventi.

The paper deals with the concept of the identity of living organisms, a concept used up until now very ambiguously. The discussion rests on the combination of two concepts, one proposed by Munzer (1993) and another derived from the considerations of Riedl (1975). The first is the proposal that the identity of living organisms depends on the properties of their elementary constituents, such as cells and tissues, and that these properties, in turn, depend on those of their DNA and RNA. It follows that the identity of a living organism remains constant or changes during life according to whether its DNA and RNA content also remains constant or changes. The second is the consideration that, during duplication of a cell population, the informational content of the population does not increase if the duplicated cells are identical (increase only of redundant DNA). On the other hand the informational content of the cell population increases if the duplicated cells are the result of a variation-selection process (increase of essential DNA). The changes of DNA and RNA content, occurring in the germinal cells during phylogenesis and in the somatic cells of the evolutionary systems during ontogenesis, lead, therefore, to the generation of new identities. Living organisms are suggested to reflect two types of identity, that of the deterministic and that of the evolutionary systems. Since the informational content of the deterministic systems (the essential DNA content) remains approximately constant during life, their identity also remains constant. The changes in the number of elementary constituents and cell volumes during the processes of hypertrophy and atrophy are accompanied only by changes in the amount of DNA (the redundant DNA). On the other hand the informational content of the evolutionary systems (the essential DNA), such as the brain-mind system, the immunological system and some receptor systems, undergo a marked increase during the ontogenic development: this leads to changes of identity of these systems. For example, in the immunological system the process of mutation and recombination of the DNA of the immunological cells leads to the generation of new proteins in the amount about 10,000 times larger than that produced through the decodification of the genome. Also the construction of the neural network, and of a number of synapses much larger than that of the neuronal cells, requires the generation of an amount of new information much larger than that contained in the genome. In short, the attribution of a double identity to living organisms reflects the simultaneous presence of systems developing either within strictly programmed limits or without programs and limits, say as closed or open projects. The difference between the two types of systems explains the different effects in the case of the transplants. The identity of the recipient of transplants is not altered in the case of transplants of a deterministic system but is so in case of transplants of evolutionary systems. There is now a widespread fear of the possibility of human cloning. It is argued that this fear is unjustified because a cloning process can never succeed in duplicating those parts which are essential for the characters of humans, namely those concerned with the properties of the evolutionary systems.

The cement of medical thought. Evolutionary emergence and downward causation.

The aetio-pathogenetic sequences and the physio-pathological patterns of diabetes, emphysema, cholera, circulatory shock and thrombosis have been analysed with respect to an evolutionary interpretation. The diseases, although reflecting alterations of processes that can always be described in physico-chemical language, occur only at the level of biological systems which reflects the decodification of genomic project: the teleonomic projects that have been developed during evolution. The concepts of evolutionary emergence and of downward causation have been used to discuss the relationship between the molecular events responsible for the initiation of the disease, and the subsequent events responsible for the aetio-pathogenesis, for the systemic disarrangement and for the additional alterations of tissues and cells independent of the initial molecular events. In diabetes the systemic disarrangement, glycosuria and hyperglycemia, reflect the evolutionary emergence of the processes regulating carbohydrate metabolism, whereas the cardiovascular and neurological alterations are effects of the systemic disarrangement by a mechanism of downward causation. The complexity of the aetio-pathogenesis and of the physio-pathological patterns of diseases is due to the generation of information during the evolution of multi-hierarchical entities. The evolutionary epistemology approach is useful to explain the behaviour of complex systems.

Adaptation and information in ontogenesis and phylogenesis. Increase of complexity and efficiency.

Adaptations during phylogenesis or ontogenesis can occur either by maintaining constant or by increasing the informational content of the organism. In the former case the increasing adaptations to external perturbation are achieved by increasing the rate of genome replication; the increased amount of DNA reflects an increase of total but not of law informational content. In the latter case the adaptations are achieved by either istructionist or evolutionary mechanism or a combination of both. Evolutionary adaptations occur during ontogenesis mainly in the brain-mind, immunological and receptor systems and involve a repertoire of receptors that are., clonally distributed, genome-conditioned and amplified by somatic mutation. Specificity and intensity of responses are achieved a posteriori as a result of natural selection of the clones. The major adaptations during phylogenesis are accompanied by increased complexity. They have been attributed to shifts, short in time and space, against the entropic drive and thus occur notwithstanding the entropic drive and the second law of thermodynamics. The alternative view, is that the generation of complexity is due to the second law of thermodynamics in its extended formulation which includes Prigogine's theorem of minimum entropy production. This view requires however that natural selection provides the biological system with structures that bring the reactions within Onsager's range. The hierarchical organization of the natural world thus reflects a stratified thermodynamic stability. As the evolutionary adaptations generate new information they may be assimilated to Maxwell demon type of processes.

[Predicibility of scientific medicine. 1. The role of chaotic and evolutionary processes]. / La predicibilità della medicina scientifica. 1. Il ruolo dei processi caotici ed evoluzionistici.

The deterministic processes, whether mechanicistic or statistic but not chaotic, have a high degree of predicibility in contrast with evolutionary processes. Following the methodological principle of C. Bernard of diseases as alterations of biochemical and physiological processes, medicine has been assigned to the area of the functional biological, i.e. to the area of the deterministic processes. However in many cases the evolution of pathological processes is hardly predictable due to many reasons. The first is the exponential evolution of many physiological processes, a reason for which the evolution tends to become chaotic. The second is that, although deterministic, frequency and distribution of diseases require a statistical approach. The third and most important reason is that a large number of pathological processes are of evolutionary nature and are therefore accompanied by the continuous generation of new information. Some examples of evolutionary processes, such the tumor trasformations and the autoimmune diseases are discussed but it is suggested that the field of evolutionary medicine will rapidly expand.

The nature of uncoupling by n-hexane, 1-hexanethiol and 1-hexanol in rat liver mitochondria.

We have analyzed the effects of n-hexane, 1-hexanethiol, and 1-hexanol on the coupled respiration of rat liver mitochondria. Incubation of mitochondria with n-hexane, 1-hexanethiol and 1-hexanol resulted in a stimulation, at low concentrations, and an inhibition, at high concentrations, of the state 4 mitochondrial respiration. Three criteria, all based on the comparison with the effect of DNP, have been used to establish whether the stimulation of respiration, at low concentrations of n-hexane, 1-hexanethiol, and 1-hexanol, depends on protonophoric mechanisms. First, the quantitative relationship between the extents of respiratory stimulation and membrane potential depression: a strong decrease of membrane potential was induced by increasing concentrations of DNP and a negligible depression by increasing concentrations of n-hexane or 1-hexanethiol. Only a slight decrease was induced by 1-hexanol. Second, the quantitative relationship between the extents of respiratory stimulation and of proton conductance increase: at equivalent rates of respiration, the enhancement of the proton conductance induced by DNP was very marked, by n-hexane and 1-hexanethiol practically negligible, and by 1-hexanol much smaller than that induced by DNP. Third, in titrations with redox inhibitors of the proton pumps, the pattern of the relationship between proton pump conductance and membrane potential was markedly different from protonophoric and non-protonophoric uncouplers: almost linear in the case of DNP, highly non-linear in the case of n-hexane, 1-hexanethiol and 1-hexanol. These three criteria support the view that n-hexane, 1-hexanethiol, and partially 1-hexanol, uncouple mitochondrial respiration by a non-protonophoric mechanism.

The nature of diseases: evolutionary, thermodynamic and historical aspects.

Physico-chemical sciences are dominated by the deterministic interpretation. Scientific medicine has generally been assigned to the area of functional biology and thence to the physico-chemical sciences. In as much as diseases are alterations of physiological processes, they share the ontological status of the latter. However, many diseases cannot be accommodated within a deterministic interpretation. First, many diseases are initiated by errors in transmission of information and followed by natural selection. These diseases, such as tumoural transformations and autoimmune processes, behave as evolutionary processes. Second, physiological processes do not cause irreversible changes while diseases may do so when not followed by restitutio ad integrum. The tendency of living organisms to maintain stationary states of great stability and minimum energy dissipation is largely due to intermolecular forces-stabilized structures, the information for which is selected during phylogenesis and decodified during ontogenesis. Diseases cause alterations of the biological structures, thereby shifting living organisms toward stationary states of lower stability and increased dissipation. The shift, reversible or irreversible, to less stable and efficient stationary states is a common thermodynamic feature of diseases. In spite of the uniqueness of their genotype, living organisms, during ontogenetic development, form spatio-temporal unrestricted classes of infinite membership. Neither stationarity nor environment-induced perturbations and consequent adaptations are sources of historicity because of the genomic programme constraint. Historicity is conferred, however, to each organism by the permanent record of such unique events as: a) the variation-selection processes occurring in the brain-mind and immunological systems; and b) irreversible alterations induced by diseases. The disease-induced changes have ontological and epistemological consequences. Since biological structures and functions are transformed into individual, historical entities, the laws of scientific medicine must be applied in clinical practice to higher levels of organization, namely to the ensembles or groups of individuals affected by the diseases.

Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c.

Hyperthyroid mitochondria show an increased Km and Vmax in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore-treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler-induced extra-respiration and membrane potential is highly non-linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.

The nature of mitochondrial respiration and discrimination between membrane and pump properties.

A new criterion is utilized for the interpretation of flow-force relationships in rat liver mitochondria. The criterion is based on the view that the nature of the relationship between the H+/O ratio and the membrane potential can be inferred from the relationship between ohmic-uncoupler-induced extra respiration and the membrane potential. Thus a linear relationship between extra respiration and membrane potential indicates unequivocally the independence of the H+/O ratio from the membrane potential and the leak nature of the resting respiration [Brand, Chien, and Diolez (1994) Biochem. J. 297, 27-29]. On the other hand, a non-linear relationship indicates that the H+/O ratio is dependent on the membrane potential. The experimental assessment of this relationship in the presence of an additional ohmic leak, however, is rendered difficult by both the uncoupler-induced depression of membrane potential and the limited range of dependence of the H+/O ratio on the membrane potential. We have selected conditions, i.e. incubation of mitochondria at low temperatures, where the extent of non-linearity is markedly increased. It appears that the nature of the resting respiration of mitochondria in vitro is markedly dependent on the temperature: at low temperatures the percentage of resting respiration due to membrane leak decreases and that due to intrinsic uncoupling of the proton pumps increases.

The effect of the protonmotive force on the redox state of mitochondrial cytochromes.

In the absence of kinetic limitations, as determined either by high substrate concentrations or by absence of respiratory chain inhibitors, we have observed that: (a) the relationship between the percentage reduction of the cytochromes and the protonmotive force is linear in the case of cytochrome c and biphasic in the case of cytochrome b, (b) the redox state of cytochrome c depends only on the membrane potential and not on the total proton motive force and (c) the alkalinization of the matrix enhances the extent of cytochrome c reduction because of the marked inhibitory effect on the cytochrome oxidase activity. Thus, although the redox states of the b, c and aa3 mitochondrial cytochromes depend on the protonmotive force, the quantitative correlation between the two parameters and the relative effects of the electrical and chemical components of the force differ among the various cytochromes.

The effect of respiration on the permeability of the mitochondrial membrane to ions.

1. The rates of cation uptake, for either organic cations such as tetrapropylammonium, TPA+, at variable tetraphenylboron concentrations, TPB-, or inorganic cations such as Mn2+, or K+ plus valinomycin, have been measured in mitochondria either respiring, under uncoupler titrations, or non-respiring, under variable K+ diffusion potentials. 2. The flow-force relationship for the respiration-coupled ion fluxes during titrations with uncouplers is almost identical to that obtained for the K(+)-diffusion driven fluxes. Similar results are obtained when TPA+ is replaced with inorganic cations, either monovalent such as K+ (+valinomycin), or divalent such as Mn2+. 3. By applying the Eyring analysis, as developed by Garlid et al. (Garlid, K.D., Beavis, A.D. and Ratkje, S.K. (1989) Biochim. Biophys. Acta 976, 109-121), from the flux-voltage relationships the values for the permeability coefficients and for the energy barriers have been obtained for the transport of the ion pair TPA(+)-TPB-, of Mn2+ and of K+ plus valinomycin, in non-respiring and in respiring, coupled and uncoupled, mitochondria. 4. The findings that the rates of respiration-coupled ion fluxes, at all values of membrane potential, are similar to the rates of the K+ diffusion potential-coupled ion fluxes and the similar pattern of the flux-voltage relationships during the titrations with uncouplers and artificial gradients indicate that the membrane permeability for ions is not modified by respiration.

Mechanism of loss of thermodynamic control in mitochondria due to hyperthyroidism and temperature.

Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.

Tracking of proton flow during transition from anaerobiosis to steady state. 2. Effect of cation uptake on the response of a hydrophobic membrane bound pH indicator.

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)-5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation efflux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of the membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported.

Tracking of proton flow during transition from anaerobiosis to steady state. 1. Response of matrix pH indicators.

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non-respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions. 2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA-untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2-3 nmol H+/mg, reduced to 1-2 nmol H+/mg in EGTA-treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6-7 nmol H+/mg, it appears that 75-90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red. 3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation. 4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.

Activation of respiration and loss of thermodynamic control in hyperthyroidism. Is it due to increased slipping in mitochondrial proton pumps?

T3 administration increases the extent of non-linearity in the flow-force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitochondria at 45 degrees C also causes an increased non-linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria.

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca2+ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca2+.

Flow-force relationships during energy transfer between mitochondrial proton pumps.

The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps.

Bulk phase proton fluxes during the generation of membrane potential in rat liver mitochondria.

The addition of oxygen to anaerobic rat liver mitochondria incubated at 15 degrees C in the absence of permeant cations produced negligible rapid H+ ejection, monitored spectroscopically with phenol red, which corresponded kinetically to the rise in delta psi, as monitored by merocyanine 540. Slow H+ translocation was observed under these conditions during the aerobic phase, the extent of which was proportional to the amount of oxygen added and the rate dependent on the rate of counter-ion movement. Measurement of H+ disappearance in the mitochondrial matrix, as monitored by neutral red, likewise showed little or no rapid H+ change in the absence of counter-ion movements. In the presence of permeant cations, the H+ disappearance in the matrix was readily measured. This observation argues against the importance of the mitochondrial outer membrane and intermembrane space in masking H+ movements. The H+ translocation required in the generation of maximal or static head delta psi was determined by following the spectral response of merocyanine to increasing oxygen additions. The amount of oxygen giving maximal Delta psi corresponds to an extrusion of 2-3 ng ions of H+ . mg of protein-1. The absence of H+ movement of near this magnitude during the development of the Delta psi argues against the Delta psi-driven backflow of H+ ions as the sole explanation of these observations.

Flux ratios and pump stoichiometries at sites II and III in liver mitochondria. Effect of slips and leaks.

Addition of bovine serum albumin to state 4 mitochondria results in a depression of the proton leak and of the resting respiration of 70 and 25%, respectively. The conductance membrane potential diagram, both in the ohmic and in the non-ohmic region, shows that in the presence of bovine serum albumin the level of ohmic conductance is lowered while that of non-ohmic conductance is increased toward higher delta psi values. The same effect is observed during operation of the different proton pumps. Addition of chloroform affects the conductance membrane potential diagram in the following manner: there is no effect in the ohmic region with all pumps, while there is an effect in the non-ohmic region either at site III or at sites II plus III but not at site II. This suggests a possible effect of chloroform at the level of the cytochrome oxidase proton pump. During titration with oligomycin of the ATPase proton pump the conductance potential diagram shows a region of non-ohmicity only in the presence but not in the absence of an ATP-regenerating system. Protonophoric uncouplers such as carbonyl cyanide p(trifluoromethoxy)phenylhydrazone and intrinsic uncouplers such as chloroform have different effects on the relationship between rates of charge translocation and of oxygen consumption, and thus on the pump stoichiometries, in that the slope of the diagram is modified by the latter but not by the former. The differential effects of protonophores and of intrinsic uncouplers on the stoichiometries have been analyzed by computer simulations and represent an additional criterion to distinguish between extrinsic and intrinsic mechanisms of uncoupling.

On the nature of the uncoupling effect of fatty acids.

The effect of palmitic acid on the electrical potential differences delta psi across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisetto et al. (1987), Biochemistry, 26, 7332-7338) delta psi decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986), Biochemistry, 25, 1747-1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the delta psi assay fails to measure the decline of delta psi in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration.