Self-Organization in Living Cells: Networks of Protein Machines and Nonequilibrium Soft Matter

Microscopic self-organization phenomena inside a living cell should not represent merely a reduced copy of self-organization in macroscopic systems. A cell is populated by active protein machines that communicate via small molecules diffusing through the cytoplasm. Mutual synchronization of machine cycles can spontaneously develop in such networks – an effect which is similar to coherent laser generation. On the other hand, an interplay between reactions, diffusion and phase transitions in biological soft matter may lead to the formation of stationary or traveling nonequilibrium nanoscale structures.     

Rapidly exchanging Ca2+ stores: Role in the control of Ca2+ homeostasis and [Ca2+] in oscillations

The rapidly exchanging intracellular calcium stores play an important role in control of cytoplasmic calcium homeostasis and in generation of intracellular calcium signals. These stores are specific intracellular compartments which are able to accumulate and release calcium in response to appropriate stimuli. Two types of stores can be distinguished in nonmuscle cells based on substances discharging these stores: (1) Ca²⁺-sensitive and (2) inositol-1,4,5-trisphosphate-sensitive intracellular depots. These two depots can be either separate intracellular compartments or a single compartment that shares both releasing mechanisms. The state of the art of our understanding of the cytoplasmic calcium release is the focus of this review.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 9–15, January–February, 1994.     

Gap junction formation and functional interaction between neonatal rat cardiocytes in culture: A correlative physiological and ultrastructural study

Summary The time course of gap junction formation and growth, following contraction synchronization of cardiac myocytes in culture, has been studied in a combined (electro)physiological and ultrastructural study. In cultures of collagenase-dissociated neonatal rat cardiocytes, pairs of spontaneously beating myocytes synchronized their contractions within one beat interval within 2–20 min after they apparently had grown into contact, 45 sec after the first synchronized beat an appreciable junctional region containing several small gap junctions was already present. In the following 30 min, neither the area of individual gap junctions nor their total area increased, 75 min after synchronization both the area of individual gap junctions and their total area had increased by a factor of 10–15 with respect to what was found in the first half hour. In the period between 75 and 300 min again no further increase in gap junctional area was found. In double voltage-clamp experiments, gap junctions between well-coupled cells behaved like ohmic conductors. In poorly coupled cells, in which the number of functional gap-junctional channels was greatly reduced, the remaining channels showed voltage-dependent gating. Their single-channel conductance was 40–50 pS. The electrophysiologically measured junctional conductance agreed well with the conductance calculated from the morphometrically determined gap-junctional area. It is concluded that a rapid initial gap junction formation occurs during the 2–20 min period prior to synchronization by assembly of functional channels from existing channel precursors already present in the cell membranes. It then takes at least another 30 min before the gap-junctional area increases possibly byde novo synthesis or by recruitment from intracellular stores or from nonjunctional membranes, a process completed in the next 45 min.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360–1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2′,7′-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 μM of cadmium acetate for 4 h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2×10⁻⁶), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC→AT transitions and decreased the frequencies of TA→AT and TA→GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC→AT transitions upon cadmium exposure.     

Stoffwechselrhythmen bei aeroben Hefen

Zusammenfassung Hefen mit aerobem Stoffwechsel zeigen nach Synchronisierung durch Aushungern rhythmische Änderungen der Glutamat-, Pyruvat-, Citratund Malat-Pools. Die Cycluslänge der Rhythmen beträgt etwa 20–30 sec. Dabei sind die Oscillationen der extramitochondrialen Pools um eine halbe Periode gegen die der intramitochondrialen Pools verschoben; diese Gegenläufigkeit ist nicht streng festgelegt.Da eine Beteiligung von Oscillationen der Atmung und Gärung ausgeschlossen werden konnte, werden Permeabilitätsänderungen der inneren Mitochondrienmembran als Ursache der Rhythmen diskutiert.Die in den vorliegenden Versuchen nachgewiesenen Rhythmen verschiedener Stoffwechselzwischenprodukte werden durch Zusatz von Kieselgel zur Zellsuspension gelöscht. Es wird angenommen, daß dabei intercellulär synchronisierende leakage-Produkte durch Adsorption aus dem Medium entfernt werden.

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Metabolic rhythms of aerobic yeasts

Summary Aerobic yeasts show rhythmic changes of glutamate, pyruvate, citrate and malate pools after synchronization by starvation. The length of each cycle is about 20–30 sec. The oscillations of extramitochondrial and intramitochondrial pools are shifted by one half period; this phase relation is not exactly fixed.Since oscillations of glycolysis and respiration are not involved, changes in the permeability of the inner mitochondrial membrane are discussed as the primary cause of the rhythms.The rhythms of the various metabolic intermediates shown in these experiments disappear when silica gel is added to the cell suspension. It is thus supposed that leakage-products mediating intercellular synchronization are withdrawn from the medium by adsorption.

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Auszung aus der gleichlautenden Dissertation der Naturwissenschaftlichen Fakultät der Technischen Universität Braunschweig 1970.     

Cooperative effect of random and time-periodic coupling strength on synchronization transitions in one-way coupled neural system: mean field approach

The cooperative effect of random coupling strength and time-periodic coupling strengh on synchronization transitions in one-way coupled neural system has been investigated by mean field approach. Results show that cooperative coupling strength (CCS) plays an active role for the enhancement of synchronization transitions. There exist an optimal frequency of CCS which makes the system display the best CCS-induced synchronization transitions, a critical frequency of CCS which can not further affect the CCS-induced synchronization transitions, and a critical amplitude of CCS which can not occur the CCS-induced synchronization transitions. Meanwhile, noise intensity plays a negative role for the CCS-induced synchronization transitions. Furthermore, it is found that the novel CCS amplitude-induced synchronization transitions and CCS frequency-induced synchronization transitions are found.     

Further studies of nerve membranes labeled with fluorescent probes

Summary By using the technique of intracellular perfusion combined with fluorescence measurements, the mode of binding of 6-p-toluidinylnaphthalene-2-sulfonate (2–6 TNS) in a squid giant axon was examined. The apparent dissociation constant for the binding sites in axons was found to be roughly 0.22mm. Out of approximately 5×10¹⁴ molecules/cm² of 2–6 TNS bound to the sites in and near the axonal membrane, roughly 2×10¹⁰ molecules/cm² are shown to contribute to a transient decrease in fluorescence during nerve excitation. By recording fluorescence signals with a polarizer and analyzer inserted in four different combinations of orientations, studies were made of the directions of the transition moments of various probe molecules relative to the longitudinal axis of the axon. Among hydrophobic probes examined, the polarization characteristics of the fluorescence signals obtained with 1–8 derivatives of aminonaphthalenesulfonate (1-8 ANS, 1-8 TNS and 1-8 AmNS) were found to be very different from those obtained with 2–6 derivatives (2-6 ANS, 2-6 TNS and 2-6 MANS). A tentative interpretation is proposed to account for this difference in physiological behavior between 1–8 and 2–6 derivatives. It is emphasized that measurements of fluorescence polarization yield significant information concerning the structure of the axonal membrane.     

Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

Applying variations of the Ovsynch protocol at the middle of the estrus cycle on reproductive performance of lactating dairy cows during summer and winter

Two modifications of the Ovsynch protocol, GnRH + TAI after PGF_2α 48 h (CO-48) or 72 h (CO-72), were compared with the original protocol (OVS: GnRH–7 d–PGF_2α–2 d–GnRH–16 h–TAI) to study their effects on reproductive performance in 785 lactating dairy cows (Holstein Friesian, Bos Taurus). Results showed that more cows (P < 0.001) returned to estrus within a week after TAI with CO-48 treatment compared with that in OVS and CO-72 treatments. Pregnancy rates were greater (P < 0.001) for the CO-72 cows than those for both OVS and CO-48 treatments and for primiparous cows compared with multiparous cows. Moreover, pregnancy rates were lower in summer compared with those in winter. Pregnancy losses for cows in both CO-48 and CO-72 were greater (P < 0.05) than that for cows in OVS treatment. Pregnancy losses were greater in summer (P < 0.001) than in winter and for multiparous cows (P < 0.001) than for primiparous cows. In conclusion, primiparous and winter-bred cows had greater pregnancy rates and fewer pregnancy losses than those of multiparous cows and summer-bred cows, respectively. Because of the presence of significant treatment, parity, and season interactions, TAI with ovulation synchronization protocols should be tailored according to the season and parity. CO-72 is recommended for primiparous cows but not for multiparous cows, and CO-48 is not recommended for synchronization. Furthermore, cows that exhibited estrus at any time were inseminated to improve pregnancy rates in ovulation synchronization protocols.     

The Mere Co-Presence: Synchronization of Autonomic Signals and Emotional Responses across Co-Present Individuals Not Engaged in Direct Interaction

Existing evidence suggests that in social contexts individuals become coupled in their emotions and behaviors. Furthermore, recent biological studies demonstrate that the physiological signals of interacting individuals become coupled as well, exhibiting temporally synchronized response patterns. However, it is yet unknown whether people can shape each other''s responses without the direct, face-to-face interaction. Here we investigated whether the convergence of physiological and emotional states can occur among “merely co-present” individuals, without direct interactional exchanges. To this end, we measured continuous autonomic signals and collected emotional responses of participants who watched emotional movies together, seated side-by-side. We found that the autonomic signals of co-present participants were idiosyncratically synchronized and that the degree of this synchronization was correlated with the convergence of their emotional responses. These findings suggest that moment-to-moment emotional transmissions, resulting in shared emotional experiences, can occur in the absence of direct communication and are mediated by autonomic synchronization.     

Non-bicarbonate intracellular pH buffering of reptilian muscle

Summary Non-bicarbonate intracellular pH buffering values of skeletal and cardiac muscles were measured for 16 species of Australian reptiles from four orders (snakes, skelctal 19–36 slykes, cardiac 9–17 slykes; lizards, skeletal 25–54 slykes, cardiac 17–19 slykes; turtles, skeletal 25–43 slykes, cardiac 11–24 slykes; crocodile, skeletal 43 slykes). Although a positive correlation between pH buffering capacity and dependence on anaerobic muscle work was found, even the highest reptilian pH buffering values were low relative to equivalent white anaerobic muscles of fish, birds, and mammals. The low non-bicarbonate intracellular pH buffering capacity of reptilian muscle arises through lower contributions from proteins (10–14 slykes), non-protein histidine (7–18 slykes) and phosphate (5–15 slykes). It is concluded that while other vertebrates depend on these intracellular buffers for regulating muscle pH during anaerobic muscle work, reptiles rely less on buffering and instead may tolerate greater pH fluctuations.Abbreviations intracellular pH buffering capacity - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - I.D. internal diameter - LDH lactate dehydrogenase     

The role of the gills in seawater adaptation inAnguilla dieffenbachii

Summary The mean serum sodium, chloride and potassium concentrations and serum osmotic pressure of freshwaterA. dieffenbachii are 140.4 mmol 1^–1,114.0 mmol 1^–1, 6.66 mmol 1^–1 and 307.7 mOsmol respectively. Gill tissue from freshwater specimens has a water content of 4234 mg g dry wt^–1 (80.9% wet wt), a chloride space of 1852 mg water g dry wt^–1 (35.2% wet wt) and an intracellular volume of 2449 mg water g dry wt (46.0% wet wt). Estimates of the intracellular sodium and potassium concentrations for the gill tissue of freshwater eels gave values of 28.9 mmol kg intracellular water^–1 and 126.5 mmol kg intracellular water^–1 respectively. On transfer of the fish to sea water serum concentrations of sodium and chloride and serum osmotic pressure show rapid initial increases followed by a more gradual decline eventually stabilising at new levels some 100 hours after transfer (Fig. 1). The serum sodium, chloride and potassium concentrations and serum osmotic pressure of seawater-adaptedA. dieffenbaehii are 162.8 mmol 1^–1, 151.0 mmol 1^–1, 6.70 mmol 1^–1 and 376.9 mOsmol respectively.On transfer to sea water the water content and chloride space of the gill tissue is reduced and the intracellular volume is initially decreased but is rapidly restored to its original value (Fig. 2, 3). At the same time intracellular sodium and potassium concentrations are increased but the latter is fairly rapidly restored to pre-transfer levels (Fig. 4).The changes in intracellular potassium concentration can be explained largely by the changes in intracellular volume but intracellular sodium concentrations remain high on transfer because of the increased serum sodium concentration. The initial increases in serum concentrations on transfer to sea water are caused partly by the removal of water and partly by the addition of sodium and chloride ions to the internal body fluids.     

Influx of Ca ion in cultured excitable cells studied by fluorescence microscope imaging technique

The spatial distribution and temporal variation of intracellular Ca ion in differentiated Neuroblastoma-Glia Hybridoma 108–15 cells (NG108–15) were investigated using a fluorescence microscope imaging technique. Fura-2 was used as a probe. Electrical current pulses of 10–20 µA were applied to axons connecting to NG cells in order to elicit the influx of Ca ion. The concentration of intracellular Ca is usually 50–80 nM in NG cells in the resting state. Upon stimulation, the Ca level increases by a factor of 2–5. The entry of Ca⁺⁺ across cell membranes is followed by intracellular diffusion and the propagation of a wave front is clearly seen in digital images. The diffusion constant was calculated to be approximately 1.66×10^–6 cm²/sec. This value is about one-fifth of the free diffusion coefficient of Ca ion in aqueous solution (7.82 × 10^–6 cm²/sec). Cd ion, at the concentration of 1–2 mM, blocks the influx of Ca as expected whereas the influx is unaffected by TTX at the concentration of 0.1 – 0.2µM.     

Glutamate Regulates the Frequency of Spontaneous Synchronized Ca<Superscript>2+</Superscript> Spikes Through Group II Metabotropic Glutamate Receptor in Cultured Mouse Cortical Networks

1. Synchronized spontaneous intracellular Ca²⁺ spikes in networked neurons are believed to play a major role in the development and plasticity of neural circuits. Glutamate-induced signals through the ionotropic glutamate receptors (iGluRs) are profoundly involved in the generation of synchronized Ca²⁺ spikes.1 2. In this study, we examined the involvement of metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons. We pharmacologically revealed that glutamate-induced signals through inclusive mGluRs decreased the frequency of Ca²⁺ spikes. Further experiments indicated that this suppressive effect on the spike frequency was mainly due to the signal through group II mGluR, inactivation of adenylate cyclase-cAMP-PKA signaling pathway. Group I mGluR had little involvement in the spike frequency.3. Taken together, glutamate generates the synchronized Ca²⁺ spikes through iGluRs and modulates simultaneously their frequency through group II mGluR–adenylate cyclase–cAMP–PKA signaling pathway in the present in vitro neural network. These results provide the evidence of the profound role of group II mGluR in the spontaneous and synchronous neural activities.     

Synchronization of spontaneous activity of skeletal muscle,autonomic nerves,and brain structures in cats

Acute and chronic experiments with recording of electrical activity of skeletal muscle, autonomic nerves, and some brain formations were performed on cats. Spectral correlation analysis showed that the spatial synchronization of electrical activity for the autonomic nerves and brain formations within the frequency range of 25–35 Hz, revealed by previous investigations, extends also to skeletal muscle. It is postulated that the presence of a widespread rhythm of 25–35 Hz is a factor facilitating the transmission of influences in the nervous system through frequency potentiation of synaptic action.Institute of Physiology, Bulgarian Academy of Sciences, Sofia. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 146–151, March–April, 1976.     

How Synchronization Protects from Noise

The functional role of synchronization has attracted much interest and debate: in particular, synchronization may allow distant sites in the brain to communicate and cooperate with each other, and therefore may play a role in temporal binding, in attention or in sensory-motor integration mechanisms. In this article, we study another role for synchronization: the so-called “collective enhancement of precision”. We argue, in a full nonlinear dynamical context, that synchronization may help protect interconnected neurons from the influence of random perturbations—intrinsic neuronal noise—which affect all neurons in the nervous system. More precisely, our main contribution is a mathematical proof that, under specific, quantified conditions, the impact of noise on individual interconnected systems and on their spatial mean can essentially be cancelled through synchronization. This property then allows reliable computations to be carried out even in the presence of significant noise (as experimentally found e.g., in retinal ganglion cells in primates). This in turn is key to obtaining meaningful downstream signals, whether in terms of precisely-timed interaction (temporal coding), population coding, or frequency coding. Similar concepts may be applicable to questions of noise and variability in systems biology.     

Excitability of the sensomotor cortex and red nucleus of rabbits at different levels of spatial synchronization of cortical potentials

To determine the excitability of the rabbit sensomotor cortex and red nucleus the animal's motor response to electrical stimulation of these structures at threshold strength was investigated. In computerized experiments the excitability of these structures was compared in situations characterized by different degrees of correlation of cortical potentials. An increase in the level of spatial synchronization of cortical potentials was shown to be accompanied by an increase in the excitability of the sensomotor cortex and red nucleus. This increase in excitability is evidently a neurophysiological mechanism of the increase in probability of appearance of an effector response to sensory stimulation when the level of spatial synchronization of cortical potentials is raised.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 19–24, January–February, 1977.     

Local and global synchronization transitions induced by time delays in small-world neuronal networks with chemical synapses

Effects of time delay on the local and global synchronization in small-world neuronal networks with chemical synapses are investigated in this paper. Numerical results show that, for both excitatory and inhibitory coupling types, the information transmission delay can always induce synchronization transitions of spiking neurons in small-world networks. In particular, regions of in-phase and out-of-phase synchronization of connected neurons emerge intermittently as the synaptic delay increases. For excitatory coupling, all transitions to spiking synchronization occur approximately at integer multiples of the firing period of individual neurons; while for inhibitory coupling, these transitions appear at the odd multiples of the half of the firing period of neurons. More importantly, the local synchronization transition is more profound than the global synchronization transition, depending on the type of coupling synapse. For excitatory synapses, the local in-phase synchronization observed for some values of the delay also occur at a global scale; while for inhibitory ones, this synchronization, observed at the local scale, disappears at a global scale. Furthermore, the small-world structure can also affect the phase synchronization of neuronal networks. It is demonstrated that increasing the rewiring probability can always improve the global synchronization of neuronal activity, but has little effect on the local synchronization of neighboring neurons.