The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

Drug recognition of DNA. Proposal for GC minor groove specific ligands: vinylexins

In a previous publication in this journal we have proposed an isolexin-like prototype of a GC minor groove specific ligand. The present paper is devoted to refinements of this prototype (increase in specificity and in DNA binding energy). It is shown that only a very limited improvement can be obtained by increasing the proton accepting capabilities of the heteroaromatic ring systems of the prototype, although these rings interact directly with the proton donating NH2 group of guanine. On the other hand a significant increase both in GC specificity and in DNA binding energy is obtained by replacing the NH linkers of the isolexin by C = C double bonds (yielding what we term "vinylexins"). Specificity is still largely conserved and the DNA binding energy is significantly increased in monocationic vinylexins, which should thus be efficient GC minor groove specific ligands. The outstanding importance for the GC specificity of the C = C linkers is evidenced by the disappearance of this specificity when these linkers are replaced by peptide bonds (peptilexins). On the other hand vinylexins with proton donating heteroaromatic rings are, as expected, AT specific. The vinylexin family may thus represent universal minor groove binding agents susceptible to bind to any given base pair sequence of DNA, following the positioning of their proton donor and proton acceptor rings. This study confirms the insufficiency of purely geometrical and/or hydrogen bonding considerations for the correct estimation of GC versus AT specificity of groove binding ligands. These can only be accounted for by taking into consideration the overall electronic properties of the interacting species and explicitly calculating the energies of complex formation including all the relevant contributions.     

Cell genealogies in a plant meristem deduced with the aid of a 'bootstrap' L-system

Abstract. The primary root meristem of maize ( Zea mays L.) contains longitudinal files of cells arranged in groups of familial descent (sisters, cousins, etc.). These groups, or packets, show ordered sequences of cell division, which are transverse with respect to the apico-basal axis of the root. The sequences have been analysed in three zones of the meristem during the course of the first four cell generations following germination. In this period, the number of cells in the packets increases from one to 16. Theoretically, there are 48 possible division pathways that lead to the eight-cell stage, and nearly 2- × 10⁶ that lead to the 16-cell stage. However, analysis shows that only a few of all the possible pathways are used in any particular zone of the root. This restriction of pathways results from inherited sequences of asymmetric cell divisions, which lead to sister cells of unequal length. All possible division pathways can be generated by deterministic 'bootstrap' L-systems which assign different life spans to sister cells of successive generations and hence specify their subsequent sequence of divisions. These systems simulate propagating patterns of cell divisions, which agree with those actually found within the growing packets that comprise the root meristem. The patterns of division are specific to cells originating in various regions of the meristem of the germinating root. The importance of such systems is that they simulate patterns of cellular proliferation where there is ancestral dependency. They can therefore be applied in other growing and proliferating systems where this is suspected.     

Prebiotic processes in planetary atmospheres

Within 40 years of experimental studies in prebiotic chemistry, most of the building blocks of the living systems have been synthesized in plausible conditions of the primitive Earth. The starting ingredients correspond to two complementary classes: volatile organics, and their non volatile oligomers. They may have been formed in the atmosphere on the primitive Earth and/or imported by extra-terrestrial sources. Organic chemistry is involved in meteorites, comets, in the giant planets and several of their satellites. Again this chemistry presents the two complementary aspects. In particular, with a dense reduced atmosphere rich in organic compounds in gas and aerosol phases, Titan appears as a natural laboratory for studying prebiotic chemistry at a planetary scale.     

Drug Recognition of DNA. Proposal for GC Minor Groove Specific Ligands: Vinylexins

Abstract

In a previous publication in this journal (ref. 14) we have proposed an isolexin-like prototype of a GC minor groove specific ligand. The present paper is devoted to refinements of this prototype (increase in specificity and in DNA binding energy). It is shown that only a very limited improvement can be obtained by increasing the proton accepting capabilities of the heteroaromatic ring systems of the prototype, although these rings interact directly with the proton donating NH₂ group of guanine. On the other hand a significant increase both in GC specificity and in DNAbinding energy is obtained by replacing the NH linkers of the isolexin by C=C double bonds (yielding what we term “vinylexins”). Specificity is still largely conserved and the DNA binding energy is significantly increased in monocationic vinylexins, which should thus be efficient GC minor groove specific ligands. The outstanding importance for the GC specificity of the C=C linkers is evidenced by the disappearance of this specificity when these linkers are replaced by peptide bonds (peptilexins). On the other hand vinylexins with proton donating heteroaromatic rings are, as expected, AT specific. The vinylexin family may thus represent universal minor groove binding agents susceptible to bind to any given base pair sequence of DNA following the positioning of their proton donor and proton acceptor rings. This study confirms the insufficiency of purely geometrical and/or hydrogen bonding considerations for the correct estimation of GC versus AT specificity of groove binding ligands. These can only be accounted for by taking into consideration the overall electronic properties of the interacting species and explicitly calculating the energies of complex formation including all the relevant contributions.     

Activated platelets form protected zones of adhesion on fibrinogen and fibronectin-coated surfaces

Leukocytes form zones of close apposition when they adhere to ligand- coated surfaces. Because plasma proteins are excluded from these contact zones, we have termed them protected zones of adhesion. To determine whether platelets form similar protected zones of adhesion, gel-filtered platelets stimulated with thrombin or ADP were allowed to adhere to fibrinogen- or fibronectin-coated surfaces. The protein- coated surfaces with platelets attached were stained with either fluorochrome-conjugated goat anti-human fibrinogen or anti-human fibronectin antibodies, or with rhodamine-conjugated polyethylene glycol polymers. Fluorescence microscopy revealed that F(ab')2 anti- fibrinogen (100 kD) did not penetrate into the contact zones between stimulated platelets and the underlying fibrinogen-coated surface, while Fab antifibrinogen (50 kD) and 10 kD polyethylene glycol readily penetrated and stained the substrate beneath the platelets. Thrombin- or ADP-stimulated platelets also formed protected zones of adhesion on fibronectin-coated surfaces. F(ab')2 anti-fibronectin and 10 kD polyethylene glycol were excluded from these adhesion zones, indicating that they are much less permeable than those formed by platelets on fibrinogen-coated surfaces. The permeability properties of protected zones of adhesion formed by stimulated platelets on surfaces coated with both fibrinogen and fibronectin were similar to the zones of adhesion formed on fibronectin alone. mAb 7E3, directed against the alpha IIb beta 3 integrin blocked the formation of protected adhesion zones between thrombin-stimulated platelets and fibrinogen or fibronectin coated surfaces. mAb C13 is directed against the alpha 5 beta 1 integrin on platelets. Stimulated platelets treated with this mAb formed protected zones of adhesion on surfaces coated with fibronectin. These protected zones were impermeable to F(ab')2 antifibronectin but were permeable to 10 kD polyethylene glycol. These results show that activated platelets form protected zones of adhesion and that the size of molecules excluded from these zones depends upon the composition of the matrix proteins to which the platelets adhere. They also show that formation of protected zones of adhesion by platelets requires alpha IIb beta 3 integrins while the permeability properties of these zones of adhesion are regulated by both alpha IIb beta 3 and alpha 5 beta 1 integrins.     

Quantum entanglement in photoactive prebiotic systems

This paper contains the review of quantum entanglement investigations in living systems, and in the quantum mechanically modelled photoactive prebiotic kernel systems. We define our modelled self-assembled supramolecular photoactive centres, composed of one or more sensitizer molecules, precursors of fatty acids and a number of water molecules, as a photoactive prebiotic kernel systems. We propose that life first emerged in the form of such minimal photoactive prebiotic kernel systems and later in the process of evolution these photoactive prebiotic kernel systems would have produced fatty acids and covered themselves with fatty acid envelopes to become the minimal cells of the Fatty Acid World. Specifically, we model self-assembling of photoactive prebiotic systems with observed quantum entanglement phenomena. We address the idea that quantum entanglement was important in the first stages of origins of life and evolution of the biospheres because simultaneously excite two prebiotic kernels in the system by appearance of two additional quantum entangled excited states, leading to faster growth and self-replication of minimal living cells. The quantum mechanically modelled possibility of synthesizing artificial self-reproducing quantum entangled prebiotic kernel systems and minimal cells also impacts the possibility of the most probable path of emergence of protocells on the Earth or elsewhere. We also examine the quantum entangled logic gates discovered in the modelled systems composed of two prebiotic kernels. Such logic gates may have application in the destruction of cancer cells or becoming building blocks of new forms of artificial cells including magnetically active ones.     

1H magnetization transfer in hydrated gluten and flour: effects of wheat aging

The interaction of water with flour or gluten in hydrated samples was investigated by proton magnetization transfer measurements. Flour and gluten from both durum and bread wheat seeds, either unaged or artificially aged over different periods of time, were investigated. Measurements were performed at several radio frequency power levels and frequency offsets, and the data were quantitatively modeled by two interacting pools, a liquid (water) and a solid (macromolecules) one. A super-Lorentzian line shape well described the magnetization of the solid pool. Magnetization transfer was found to be more efficient for flour with respect to gluten samples, in agreement with their hydrophilic/hydrophobic behavior. The aging treatment of seeds resulted in a minor degree of interaction between macromolecules and water.     

Endopeptidases from Plasmodium knowlesi

Extracts of rhesus monkey erythrocytes infected with Plasmodium knowlesi were fractionated by polyacrylamide gel electrophoresis (PAGE) and several zones of endopeptidase activity were demonstrated by an imprint-digest method. The enzymes were active only under acid conditions; activity was detected at pH 3.2 but not between pH 6.4 and 8.9 using haemoglobin, albumin or erythrocyte lysate as the substrate. Optimized PAGE conditions separated highly active parasite enzymes with Rf values of 73, 63 and 53 (+/- 7%), as well as a red cell endopeptidase, Rf44. Of two other minor bands of activity, one was associated with platelets.     

Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a

We investigated the biochemical phenotype of the mtDNA T8993G point mutation in the ATPase 6 gene, associated with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in three patients from two unrelated families. All three carried >80% mutant genome in platelets and were manifesting clinically various degrees of the NARP phenotype. Coupled submitochondrial particles prepared from platelets capable of succinate-sustained ATP synthesis were studied using very sensitive and rapid luminometric and fluorescence methods. A sharp decrease (>95%) in the succinate-sustained ATP synthesis rate of the particles was found, but both the ATP hydrolysis rate and ATP-driven proton translocation (when the protons flow from the matrix to the cytosol) were minimally affected. The T8993G mutation changes the highly conserved residue Leu(156) to Arg in the ATPase 6 subunit (subunit a). This subunit, together with subunit c, is thought to cooperatively catalyze proton translocation and rotate, one with respect to the other, during the catalytic cycle of the F(1)F(0) complex. Our results suggest that the T8993G mutation induces a structural defect in human F(1)F(0)-ATPase that causes a severe impairment of ATP synthesis. This is possibly due to a defect in either the vectorial proton transport from the cytosol to the mitochondrial matrix or the coupling of proton flow through F(0) to ATP synthesis in F(1). Whatever mechanism is involved, this leads to impaired ATP synthesis. On the other hand, ATP hydrolysis that involves proton flow from the matrix to the cytosol is essentially unaffected.     

Emergence of homochirality in far-from-equilibrium systems: mechanisms and role in prebiotic chemistry

Since the model proposed by Frank (Frank FC, Biochem Biophys Acta 1953;11:459-463), several alternative models have been developed to explain how an asymmetric non-racemic steady state can be reached by a chirally symmetric chemical reactive system. This paper explains how a stable non-racemic regime can be obtained as a symmetry breaking occurring in a far-from-equilibrium reactive system initiated with an initial imbalance. Departing from the variations around the original Frank's model that are commonly described in the literature, i.e. open-flow systems of direct autocatalytic reactions, we discuss recent developments emphasizing both an active recycling of components and an autocatalytic network of simple reactions. We will present our APED model as the most natural realization of such thermodynamic openness and non-equilibrium, of recycling and of network autocatalysis, each of these in prebiotic conditions. The different experimental and theoretical models in the literature will be classified according to mechanism. The place and role of such self-structured networks responsible for the presence of homochirality in the primitive Earth will be detailed.     

On the Spectrum of Prebiotic Chemical Systems

We reexamine Eigen’s paradox using the asymptotic limit theorems of information theory. Applying the homology between information source uncertainty and free energy density, under rate distortion constraints, the error catastrophe emerges as the lowest energy state for simple prebiotic systems without error correction. Invoking the usual compartmentalization – i.e., ‘vesicles’ – and using a Red Queen argument, suggests that information crosstalk between two or more properly interacting structures can initiate a coevolutionary dynamic having at least two quasi-stable states. The first is a low energy realm near the error threshold, and, depending on available energy, the second can approach zero error as a limit. A large deviations argument produces jet-like global transitions which, over sufficient time, may enable shifts between the many quasi-stable modes available to more complicated structures, ‘locking in’ to some subset of the various possible low error rate chemical systems, which become subject to development by selection and chance extinction. Energy availability, according to the model, is thus a powerful necessary condition for low error rate replication, suggesting that some fundamental prebiotic ecosystem transformation entrained reproductive fidelity. This work, then, supports speculation that our RNA/DNA world may indeed be only the chance result of a very broad prebiotic evolutionary phenomenon. Processes in vitro, or ex planeta, might have other outcomes.     

High‐Output Triboelectric Nanogenerator Based on Dual Inductive and Resonance Effects‐Controlled Highly Transparent Polyimide for Self‐Powered Sensor Network Systems

High‐output triboelectric nanogenerators (TENGs) are demonstrated based on polyimide (PI)‐based polymers by introducing functionalities (e.g., electron‐withdrawing and electron‐donating groups) into the backbone. The TENG based on 6FDA‐APS PI, possessing the most negative electrostatic potential and the low‐lying lowest unoccupied molecular orbital level, produces the highest effective charge density of about 860 µC m^?2 in practical working conditions with the ion injection process. This may be ascribed to the excellent charge‐retention characteristics as well as the enhanced charge transfer capability, which increases the output power by 7 times compared to the commercially available Kapton film‐based TENG. Finally, a 6FDA‐APS‐driven sensor network system is demonstrated, providing the identity of three gases (H₂, CO, and NO₂) by illuminating the light‐emitting diodes within several seconds.     

Fluorescence energy transfer reveals microdomain formation at physiological temperatures in lipid mixtures modeling the outer leaflet of the plasma membrane

An approach is described using fluorescence resonance energy transfer (FRET) to detect inhomogeneity in lipid organization, on distance scales of the order of tens of nanometers or greater, in lipid bilayers. This approach compares the efficiency of energy transfer between two matched fluorescent lipid donors, differing in their affinities for ordered versus disordered regions of the bilayer, and an acceptor lipid that distributes preferentially into disordered regions. Inhomogeneities in bilayer organization, on spatial scales of tens of nanometers or greater, are detected as a marked difference in the efficiencies of quenching of fluorescence of the two donor species by the acceptor. Using a novel pair of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled tetraacyl lipids as donor species with a rhodaminyl-labeled acceptor, this strategy faithfully reports homo- versus inhomogeneous mixing in each of several lipid bilayer systems whose organization on the FRET distance scale can be predicted from previous findings. Interestingly, however, the present FRET method reports clear evidence of inhomogeneity in the organization of mixtures combining sphingomyelin or saturated phospholipids with unsaturated phospholipids and physiological proportions of cholesterol, even at physiological temperatures where these systems have been reported to appear homogeneous by fluorescence microscopy. These results indicate that under physiological conditions, lipid mixtures mimicking the lipid composition of the outer leaflet of the plasma membrane can form domains on a spatial scale comparable to that inferred for the dimensions of lipid rafts in biological membranes.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

Phosphorus in prebiotic chemistry

The prebiotic synthesis of phosphorus-containing compounds-such as nucleotides and polynucleotides-would require both a geologically plausible source of the element and pathways for its incorporation into chemical systems on the primitive Earth. The mineral apatite, which is the only significant source of phosphate on Earth, has long been thought to be problematical in this respect due to its low solubility and reactivity. However, in the last decade or so, at least two pathways have been demonstrated which would circumvent these perceived problems. In addition, recent results would seem to suggest an additional, extraterrestrial source of reactive phosphorus. It appears that the 'phosphorus problem' is no longer the stumbling block which it was once thought to be.     

Inversion Concept of the Origin of Life

The essence of the inversion concept of the origin of life can be narrowed down to the following theses: 1) thermodynamic inversion is the key transformation of prebiotic microsystems leading to their transition into primary forms of life; 2) this transformation might occur only in the microsystems oscillating around the bifurcation point under far-from-equilibrium conditions. The transformation consists in the inversion of the balance "free energy contribution / entropy contribution", from negative to positive values. At the inversion moment the microsystem radically reorganizes in accordance with the new negentropy (i.e. biological) way of organization. According to this approach, the origin-of-life process on the early Earth took place in the fluctuating hydrothermal medium. The process occurred in two successive stages: a) spontaneous self-assembly of initial three-dimensional prebiotic microsystems composed mainly of hydrocarbons, lipids and simple amino acids, or their precursors, within the temperature interval of 100-300°C (prebiotic stage); b) non-spontaneous synthesis of sugars, ATP and nucleic acids started at the inversion moment under the temperature 70-100°C (biotic stage). Macro- and microfluctuations of thermodynamic and physico-chemical parameters able to sustain this way of chemical conversion have been detected in several contemporary hydrothermal systems. A minimal self-sufficient unit of life on the early Earth was a community of simplest microorganisms (not a separate microorganism).     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Yolk platelets in Xenopus oocytes maintain an acidic internal pH which may be essential for sodium accumulation

Yolk platelets constitute an embryonic endocytic compartment that stores maternally synthesized nutrients. The pH of Xenopus yolk platelets, measured by photometry on whole oocytes which had endocytosed FITC-vitellogenin, was found to be acidic (around pH 5.6). Experiments on digitonin-permeabilized oocytes showed that acidification was due to the activity of an NEM- and bafilomycin A1- sensitive vacuolar proton-ATPase. Proton pumping required chloride, but was not influenced by potassium or sodium. Passive proton leakage was slow, probably due to the buffer capacity of the yolk, and was dependent on the presence of cytoplasmic monovalent cations. In particular, sodium could drive proton efflux through an amiloride- sensitive Na+/H+ exchanger. 8-Bromo-cyclic-AMP was found to increase acidification, suggesting that pH can be regulated by intracellular second messengers. The moderately acidic pH does not promote degradation of the yolk platelets, which in oocytes are stable for weeks, but it is likely to be required to maintain the integrity of these organelles. Furthermore, the pH gradient created by the proton pump, when coupled with the Na+/H+ exchanger, is probably responsible for the accumulation and storage of sodium into the yolk platelets during oogenesis.     

Transmembrane distribution of lipophilic cations in response to an electrochemical potential in reconstituted cytochromec oxidase vesicles and in vesicles exhibiting a potassium ion diffusion potential

It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochromec oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.