A method was developed for stabilizing mitotic chromosomes. Light irradiation of permeabilized cells in a low concentration of ethidium bromide made chromatin resistant to high salt concentrations and decondensing buffer. This resistance was abolished by proteinase treatment, but not by DNase or RNase treatment. In photostabilized and extracted chromosomes, chromatin appeared as thick fibers with discrete high electron density regions. These stabilized structures might correspond to the higher-level structures (chromonemata) observed in native chromatin. Moreover, the electron density was higher in the centromeric regions than the chromosome arm material. Thus, the method allows chromatin substructures (chromonemata and centromeric heterochromatin) to be stabilized inside mitotic chromosomes.

Preparations of new low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat Fagopyrum esculentum seeds by chromatography of seed extracts on trypsin-Sepharose 4B, Mono-Q and Mono-S ion-exchangers. Their molecular masses, determined by mass spectrometry, were equal to 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c) and 6031 daltons (BWI-4c). All inhibitors possessed high pH-stability in the pH range 2-12 and thermostability. In addition to trypsin, BWI-3c and BWI-4c inhibitors inhibited chymotrypsin and subtilisin-like proteases. The inhibition constants (Ki) for trypsin, chymotrypsin and subtilisin by the studied inhibitors were determined. The N-terminal sequences of all inhibitors were established: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues) and BWI-4c (20 residues). According to the physicochemical properties and N-terminal amino acid sequences, buckwheat seed protease inhibitors BWI-3c and BWI-4c are suggested to belong to the potato proteinase inhibitor I family.

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.

Cells of cultured line XL2 (Xenopus laevis) were synchronized by a combine effect of serum deprivation, aphidicolin, nocodazole and ALLN treatments. Four fractions were prepared, with maximum percentage of cells being in G1, S and G2 phases of cell cycle, and in mitosis, respectively. Comparative levels of six different proteins (β-tubulin, DNA topoisomerase IIa, Xenopus aurora A kinase pEg2, kinesin-like motor protein X1Eg5, and two members of condensis family proteins pEg7 (XCAP D2) and XCAP E were detected by quantitative Western blot analysis of these fractions. We used a new method of mathematic processing of data that commonly provides a possibility to calculate a comparative quantity of proteins in hypothetically "clean" fraction composed of cells being in the same phase of the cell cycle. This method makes it possible to use even partly synchronized cell cultures for analysis of changes in protein quantity, provided a precede determination of cell population composition is made.

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.

Dissemination of neoplastic cells from the primary tumor (invasion and metastasis) is a fundamentally dangerous step in multistage carcinogenesis. Recent evidence suggests that Rho GTPase-mediated signaling is linked to dissemination of cells from several different types of human tumors. The Rho family of proteins is typically associated with the regulation of cytoskeletal activity, including actin assembly, microtubule dynamics, and myosin II-dependent contractility of the actin-rich cortex. We examined the effect of overexpression of constitutively active RhoA on islands and monolayers of epithelial cells. Although newly plated cells initially formed small spread islands, there was also a significant population of cells that detached from the substrate, floated in the medium, and then could reattach to the substrate to form new colonies. Detachment of cells from transfected epithelial islands or monolayers occurred in correlation to the plane of cytokinesis after misorientation of the mitotic spindle axis. We suggest that these alterations result from Rho-induced increase of contractility of the cortex of dividing cells, which, during cytokinesis, produces a cell that has budded out of an existing layer of cells. Cell division-mediated detachment of cells from tissue structures may be an important mechanism of tumor dissemination and metastasis.



II. BIOENERGETICS AND PHOTOSYNTHESIS

Apoptosis was observed in the coleoptile and initial leaf in 5-8-day-old wheat seedlings grown under normal daylight. Apoptosis is an obligatory event in early wheat plant ontogenesis, and it is characterized by cytoplasmic structural reorganization and fragmentation, in particular, with the appearance in vacuoles of specific vesicles containing intact organelles, chromatin condensation and margination in the nucleus, and internucleosomal fragmentation of nuclear DNA. The earliest signs of programmed cell death (PCD) were observed in the cytoplasm, but the elements of apoptotic degradation in the nucleus appeared later. Nuclear DNA fragmentation was detected after chromatin condensation and the appearance in vacuoles of specific vesicles containing mitochondria. Two PCD varieties were observed in the initial leaf of 5-day-old seedlings grown under normal daylight: a proper apoptosis and vacuolar collapse. On the contrary, PCD in coleoptiles under various growing (light) conditions and in the initial leaf of etiolated seedlings is only a classical plant apoptosis. Therefore, various tissue-specific and light-dependent PCD forms do exist in plants. Amounts of O₂*- and H₂O₂ evolved by seedlings grown under normal daylight are less than that evolved by etiolated seedlings. The amount of H₂O₂ formed in the presence of sodium salicylate or azide by seedlings grown under normal daylight was increased. Contrary to etiolated seedlings, the antioxidant BHT (ionol) did not inhibit O₂*- formation and apoptosis and it had no influence on ontogenesis in the seedlings grown under normal daylight. Thus, in plants grown under the normal light regime the powerful system controlling the balance between formation and inactivation of reactive oxygen species (ROS) does exist and it effectively functions. This system is responsible for maintenance of cell homeostasis, and it regulates the crucial ROS level controlling plant growth and development. In etiolated plants, this system seems to be absent, or it is much less effective.

Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (τ) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in τ is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of τ. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in τ was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.

Properties of Klebsiella pneumoniae respiratory chain enzymes catalyzing NADH oxidation have been studied. Using constructed K. pneumoniae mutant strains, it was shown that three enzymes belonging to different families of NADH:quinone oxidoreductases operate in this bacterium. The NDH-2-type enzyme is not coupled with energy conservation, the NDH-1-type enzyme is a primary proton pump, and the NQR-type enzyme is homologous to the sodium-motive NADH dehydrogenase of Vibrio and is shown to be a primary Na⁺ pump. It is concluded that the NQR-type enzyme, not the NDH-1-type enzyme, catalyzes sodium-dependent NADH oxidation in K. pneumoniae.

The enzyme content of the mitochondrial respiratory chain was investigated in the heat-producing plant Arum orientale. It is shown that mitochondria isolated from thermogenic tissues of this plant (with respect to non-thermogenic tissues of A. orientale or to Zea mays) demonstrate significantly elevated levels of activities of two non-coupled NADH dehydrogenases oxidizing intramitochondrial and cytoplasmic NADH pools. It is postulated that operation of a completely non-coupled respiratory chain consisting of non-coupled NADH:quinone oxidoreductases and cyanide-resistant alternative quinol-oxidase is the main mechanism of heat production in thermogenic plants.

The atomic structures of photosynthetic reaction centers of two species of purple bacteria and two photosystems 2 of cyanobacteria were resolved in the late last century. In this work I put forward the idea that of the huge body of data available thus far, only three structural factors are responsible for the unique function of conversion of physical energy of electronic excitation into electrochemical energy of separated opposite charges in reaction centers at least in purple bacteria and, perhaps, in other photosynthetic organisms.


Literary evidence for a link between mutations in genes encoding respiratory chain components and human disorders is reviewed with particular emphasis on defects in respiratory complexes III and IV and their assembly factors. To date, mutations in genes encoding cytochrome band QP-C structural subunits of cytochrome bc1 complex; the BCS1L assembly factor for the bc1 complex; structural subunits I-III of cytochrome c oxidase; as well as the SURF-1, COX10, SCO1, and SCO2 assembly factors for cytochrome c oxidase, have been reported. These mutations are responsible for different neuromuscular and non-neuromuscular human diseases.

Cytochrome bd is a prokaryotic terminal oxidase catalyzing O₂ reduction to H₂O. The oxygen-reducing site has been proposed to contain two hemes, d and b595, the latter presumably replacing functionally CuB of heme-copper oxidases. We show that NO, in competition with O₂, rapidly and potently (Ki = 100 ± 34 nM at approximately 70 μM O₂) inhibits cytochrome bd isolated from Escherichia coli and Azotobacter vinelandii in turnover, inhibition being quickly and fully reverted upon NO depletion. Under anaerobic reducing conditions, neither of the two enzymes reveals NO reductase activity, which is proposed to be associated with CuB in heme-copper oxidases.

Frequency domain spectra of the photosystem I (PS1) of Synechococcus elongatus are measured in a wide temperature range and explained in an exciton model based on the recently determined X-ray crystal structure. Using the known spatial positions and orientations of the chlorophylls (Chls) the dipole-dipole couplings between the chromophores are calculated. In contrast, the Chl Qy site energies are determined by a simultaneous fit of low-temperature absorption, linear dichroism, and circular dichroism spectra. The best fit is achieved by an evolutionary algorithm after assigning some chromophores to the red-most states. Furthermore, a microscopically founded homogeneous line width is included and the influence of inhomogeneous broadening is discussed. To confirm the quality of the resulting PS1 model, time-dependent fluorescence spectra are calculated, showing a good agreement with recent experimental results.

Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer. Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps. Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons. By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier. The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H⁺/OH^- ions of  120 meV. We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine.

Based on the available experimental data, we developed a kinetic model of the catalytic cycle of imidazologlycerol phosphate synthetase from Escherichia coli accounting for the synthetase and glutaminase activities of the enzyme. The rate equations describing synthetase and glutaminase activities of imidazologlycerol phosphate synthetase were derived from this catalytic cycle. Using the literature data, we evaluated all kinetic parameters of the rate equations characterizing individually synthetase and glutaminase activities as well as the contribution of each activity depending on concentration of the substrates, products, and effectors. As shown, in the presence of 5 .phosphoribosylformimino 5 aminoimidazolo 4 carboxamideribonucleotide (ProFAR) and imidazologlycerol phosphate (IGP)glutaminase activity dominates over synthetase activity at sufficiently low concentrations of 5 .phosphoribulosylformimino 5 aminoimidazolo 4 carboxamideribonucleotide (PRFAR). Increased PRFAR concentrations resulted in decreased contribution of glutaminase activity and, consequently, increased the contribution of synthetase activity in the enzyme functioning.

The membrane portion of F0F1-ATP synthase, F0, translocates protons by a rotary mechanism. Proton conduction by F0 was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F0. The current-voltage relationship of F0 was linear from 7 to 70 mV. The current was extremely proton-specific (>107) and varied only slightly ( threefold) from pH 6 to 10. The maximum conductance was  10 fS at pH 8, equivalent to 6240 H⁺ s–1 at 100-mV driving force, which is an order-of-magnitude greater than of coupled F0F1. There was no voltage-gating of F0 even at low voltage, and proton translocation could be driven by  pH alone, without voltage. The reported voltage gating in F0F1 is thus attributable to the interaction of F0 with F1 but not to F0 proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of  6 and  10 to these relays, and placed them close to the membrane/electrolyte interface.

In accordance with our concept of rigorous optimization of photosynthetic machinery by a functional criterion, this series of papers continues purposeful search in natural photosynthetic units (PSU) for the basic principles of their organization that we predicted theoretically for optimal model light-harvesting systems. This approach allowed us to determine the basic principles for the organization of a PSU of any fixed size. This series of papers deals with the problem of structure optimization for light-harvesting antennae of variable size controlled in vivo by the light intensity during the growth of organisms, which accentuates the problem of antenna structure optimization because optimization requirements become more stringent as the PSU increases in size. In this work, using mathematical modeling for the functioning of natural PSUs, we have shown that the aggregation of pigments of model light-harvesting antenna, being one of universal optimizing factors, furthermore allows controlling the antenna efficiency if the extent of pigment aggregation is a variable parameter. In this case, the efficiency of antenna increases with the size of the elementary antenna aggregate, thus ensuring the high efficiency of the PSU irrespective of its size; i.e., variation in the extent of pigment aggregation controlled by the size of light-harvesting antenna is biologically expedient.

Oxidative stress has been implicated in neuronal death caused by cerebral ischemia or some neurologic disorders. Chemical hypoxia (term defining the simulation by using respiratory inhibitors) chosen as in vitro ischemic model, was induced in primary cultures of rat cerebellar granule neurons by inhibitors of mitochondrial electron transport such as rotenone or paraquat (complex I), 3-nitropropionic acid (3-NPA, complex II), antimycin A (complex III), or sodium azide (complex IV). All compounds caused neuronal death determined by trypan blue staining and MTT-test. On the other hand, neurotoxicity of rotenone and paraquat but not of 3-NPA, antimycin or azide was significantly abolished by menadione (vitamin K3, 2-methyl-1,4-naphthoquinone). This neuroprotective effect of menadione was associated with a decrease of rotenone-induced free radical production.

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H₂O₂-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3ß (GSK-3ß). Alternatively, receptor tyrosine kinase or certain G protein–coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3ß, via protein kinase B/Akt and mTOR/p70s6k pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3ß on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

Correspondence of phases of electrogenesis, photocycle transitions, and proton transfer with the proton transport ing groups of bacteriorhodopsin was studied. The structure of bacteriorhodopsin was considered by the file 1c3w and projections of sites of the proton movement pathway onto the normal to the purple membrane were measured. The dielectric permeability of the terminal site of the semichannel Schiff base >external surface of the purple membrane was noticeably higher than in the center of the membrane.

Acidification of a high phosphate incubation medium from pH 7.4 to 6.5 promotes increase in rates of succinate oxidation and exogenous NADH oxidation via external (rotenone- and myxothiazol-resistant) pathway by factors 2 and 2.3 respectively. Cyclosporin A prevents these effects. To measure the cytochrome c release, mitochondrial cytochrome c concentration was calculated from absorption spectrum of α-band of cytochromes c+c1. The cytochrome c release is shown to be equal to 27±4%, 40±12%, 70±5% at pH 7.4, 7.0, 6.5, respectively, the last value being reduced by cyclosporin A to 10±3%. Immunoblot method gives the similar results. It is concluded that acidification of the high phosphate medium induces release of a large part of the cytochrome c pool from liver mitochondria due to opening the Ca²⁺-dependent cyclosporin A-sensitive permeability transition pore and subsequent high amplitude swelling.

The dark reduction of photooxidized bacteriochlorophyll (P⁺) by photoreduced secondary quinone acceptor (QB–) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied.The kinetics of the dark reduction of P⁺ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium. In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P⁺ on duration of light was not observed. Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain. It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site. At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB .

The effects of specific inhibitors of respiratory chain, F0F1ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F1-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.

The inhibitors of oxidative phosphorylation induced fragmentation of mitochondria without any signs of apoptosis in CV-1 and HeLa cells. Prolonged treatment with the uncouplers (alone or in combination with the inhibitors of respiration) caused perinuclear clusterization of mitochondria, followed by their selective elimination. The fraction of mitochondria-depleted cells remained viable.

Independent experimental and theoretical evaluation was performed for the adequacy of our previously proposed general molecular model of the structural organization of light-harvesting pigments in chlorosomal bacteriochlorophyll (BChl)  /d/e-containing superantennae of different green bacteria. Measurement of the temperature dependence of steady-state fluorescence spectra of BChl c was accomplished in intact cells of a photosynthetic green bacterium Chloroflexus aurantiacus; this allows in vivo determination of the structure of exciton levels of BChl c oligomers in this natural antenna. Experimental data confirm our model of organization of oligomeric pigments in chlorosomal BChl c antenna of green bacterium Chloroflexus aurantiacus. This model implies that the unit building block of the antenna is a cylindrical assembly containing six excitonically coupled linear pigment chains, whose exciton structure with intense upper levels provides for the optimal spectral properties of the light-harvesting antenna.

Escherichia coli proteins YegE and YaiC contain N-terminal integral membrane regions, followed by the putative diguanylate cyclase (GGDEF, DUF1) domains. The membrane domains of these proteins, named MASE1 (membrane-associated sensor) and MASE2, respectively, were found in other bacterial signaling proteins, such as histidine kinases (MASE1) and an adenylate cyclase (MASE2). Although the nature of the signals sensed by MASE1 and MASE2 is still unknown, MASE1-containing receptors appear to play important roles in bacteria, including iron and/or oxygen sensing by hemerythrine-containing proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris.

We have modeled energy-transfer dynamics in the peripheral plant light-harvesting complex LHCII using both standard Redfield theory and its modification for the case of strong exciton-phonon coupling (Zhang, W. M.; Meier, T.; Chernyak, V.; Mukamel, S. J. Chem. Phys. 1998, 108, 7763). A quantitative simultaneous fit of the absorption (OD), linear dichroism (LD), steady-state fluorescence (FL) spectra at 7-293 K, and transient absorption (TA) kinetics at 77 and 293 K has been obtained using the experimental exciton-phonon spectral density to model the temperature-dependent line shape. We use configurations of the antenna (i.e., chlorophyll (Chl) a/b identities, orientations, and site energies) close to those proposed in our previous study (Novoderezhkin, V.; Salverda, J. M.; van Amerongen, H.; van Grondelle, R. J. Phys. Chem. B 2003, 107, 1893). These configurations have been further adjusted from the fit with the modified Redfield approach. The new (adjusted) models allow a better quantitative explanation of the spectral shapes. A combination of fast (femtosecond) interband energy transfer and slow (picosecond) intraband equilibration can be better reproduced as well. We paid special attention to unravel the origins of the slow components preliminarily assigned to localized states in the previous work. These "bottleneck" states have been directly visualized in this study via selective femtosecond excitation and probing at different wavelengths. In our modeling, these states are determined by two or three (depending on the model) monomeric Chls a or b shifted to the spectral region of 655-670 nm between the main absorption peaks of Chl b (650 nm) and Chl a (675 nm). In all configurations we have found these energy-shifted Chls to be bound at mixed sites (i.e., A3, A6, A7, or B3). Experiments and self-consistent modeling using the modified Redfield theory allow us to explore the participation of these states in the overall energy-transfer dynamics. This has led to a more complete and physically adequate model for the energy-transfer dynamics in LHCII.

Primary charge separation dynamics is modeled in the pheophytin-modified Rhodobacter sphaeroides R-26 reaction center (RC). To explain the observed spectral evolution, it is assumed that the process is coupled to coherent nuclear motion. A density matrix equation with the Redfield relaxation superoperator is used for simulation of the electron-vibrational dynamics and its spectral signatures. The model includes two diabatic states, i.e., an excited state P* of the primary donor (i.e., special pair, P), and a charge-transfer state (P⁺B^-, which is the primary photoproduct in the pheophytin-modified RC). The strong coupling of these states with two collective nuclear modes is supposed. The mixing of diabatic states (with different displacements along each of the two nuclear coordinates) results in a complicated potential surface that determines the dynamics of the excited-state wave packet. The coupled nuclear and charge-transfer dynamics is calculated in the basis of vibronic eigenstates obtained by numerical diagonalization of the electron-vibrational Hamiltonian. The third-order nonlinear response associated with excited-state dynamics is calculated, including the P*  P stimulated emission (SE) and the P⁺B^-  P⁺(B^-)* excited-state absorption (ESA). The model allowed us to obtain a quantitative fit of the experimental kinetics of the SE near 900-950 nm and the ESA in the 1020-nm region of the pheophytin-modified Rhodobacter sphaeroides R-26 RC (Yakovlev, A. G.; Shkuropatov, A. Ya.; Shuvalov, V. A. FEBS Lett. 2000, 466, 209). By use of the parameters adjusted from the fit, we have obtained a direct visualization of the electron-vibrational wave packet evolution, including the surface-crossing dynamics superimposed with oscillatory motion along two reaction coordinates in the P* and P⁺B^- states. It is concluded that nonequilibrated vibrational modes involved in electron-transfer play an important role in photoproduct formation in bacterial RC. We found that the specific configuration of two vibrational coordinates (obtained from the modeling) determines high efficiency of charge separation both for coherent and noncoherent excitation.

Natural uncouplers of oxidative phosphorylation, long-chain non-esterified fatty acids, cause uncoupling in the alkalo- and halotolerant bacterium Bacillus pseudofirmus FTU. The uncoupling effect in the bacterial cells was manifested as decrease of membrane potential and increase of respiratory activity. The membrane potential decrease was detected only in bacterial cells exhausted by their endogenous substrates. In proteoliposomes containing reconstituted bacterial cytochrome c oxidase, fatty acids caused a "mild" uncoupling effect by reducing membrane potential only at low rate of membrane potential generation. "Free respiration" induced by the "mild" uncouplers, the fatty acids, can be considered as possible mechanism responsible for adaptation of the bacteria to a constantly changed environment.

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo а oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Δψ in the ferryl-oxo а oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (τ approximately 15 μs), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (τ approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D₂O. With the wild type oxidase, such a high H₂O/D₂O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).

To demonstrate that an uncoupling of respiration and phosphorylation, measured in vitro, reflects an in vivo situation, we badly need in vivo measurements of some uncoupling-linked parameters. The importance of this assertion is illustrated by studies of Barja and co-workers. A lower rate of H₂O₂ production by mitochondria isolated from long-lived birds compared with short-lived mammals of the same body weight (see publications by Barja's and Sohal's groups) could be explained by (i) an in vivo difference or (ii) an in vitro artefact. In both cases, the reason for lower H₂O₂ production may well be the same, i.e. a mild uncoupling of respiration in avian mitochondria showing lowered respiratory control. Again, this should be due to an in vivo operation of some bird-specific natural uncouplers (the first case) or stronger in vitro damage to the avian mitochondria during their isolation and incubation (the second). The latter possibility seemed more probable when Barja and co-workers revealed that the level of antioxidants in birds is lower than in mammals. However, further studies by the same group showed that the degree of unsaturation of fatty acids in birds is lower than in mammals, indicating a greater resistance of avian mitochondria to oxidative damage in vitro. Indeed, it was found that lipid peroxidation in isolated avian mitochondria occurs at a much lower rate than in mammals. More importantly, the in vivo level of peroxidation of lipids and proteins appears to be lower in birds than in mammals. Thus, it seems probable that longer lifespan of birds really does correlate with a slower rate of production of H₂O₂ by mitochondria in vivo.

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of beads-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called 'mitoptosis'). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles ('cargo boats' transporting some apoptotic proteins to their nuclear targets).

The channel activity of colicin E1 was studied in planar lipid bilayers and liposomes. Colicin E1 pore-forming activity was found to depend on the curvature of the lipid bilayer, as judged by the effect on channel activity of curvature-modulating agents. In particular, the colicin-induced trans-membrane current was augmented by lysophosphatidylcholine and reduced by oleic acid, agents promoting positive and negative membrane curvature, respectively. The data obtained imply direct involvement of lipids in the formation of colicin E1-induced pore walls. It is inferred that the toroidal pore model previously validated for small antimicrobial peptides is applicable to colicin E1, a large protein that contains ten α-helices in its pore-forming domain.

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.

The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 ± 0.2) 105 M-1 s-1 at low ionic strength. Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The role of Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role. This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair.


VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator (ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.

The mechanism of Bax-dependent cytochrome c release is still controversial and may also depend on the actual localisation of cytochrome c: (i) we studied the distribution of cytochrome c in sub-fractions of rat kidney mitochondria and found that 10-20% of the total cytochrome c was associated at the peripheral inner membrane and to some extent organised in the contact sites. (ii) Cytochrome c concentrations in the contact site fractions varied related to surface bound hexokinase activity. It decreased upon reduction of contact sites by glycerol or specific dissociation of the VDAC-ANT complexes by bongkrekate, whereas it increased upon induction of contacts by dextran or association of VDAC-ANT complexes by atractyloside. (iii) The outer membrane pore (VDAC) acquires high capacity for hexokinase binding by interacting with the ANT. Thus, surface-attached hexokinase protein indicated the frequency of VDAC-ANT complexes and the correlation between hexokinase activity and cytochrome c suggested association of the latter to the complexes. (iv) Substances affecting exclusively the structure of either hexokinase (glucose-6P) or cytochrome c (borate) led to a decrease only of the effected protein without changing the concentration of other contact site constituents. (v) Hexokinase was furthermore used as a tool to isolate the contact site forming complex of outer membrane VDAC and inner membrane ANT from Triton-dissolved membranes. Cytochrome c remained attached to the hexokinase VDAC-ANT complexes that were reconstituted in phospholipid vesicles. (vi) The vesicles were loaded with malate and BaxDeltaC released the endogenous cytochrome c from the reconstituted complexes without forming unspecific pores for malate. BaxDeltaC targeted a cytochrome c fraction associated at the VDAC-ANT complex. The cytochrome c organisation was dependent on the actual structure of VDAC and ANT. Thus, the BaxDeltaC effect was suppressed either by hexokinase utilising glucose and ATP or by bongkrekic acid both influencing the pore and ANT structure.

The present series of papers is part of an integrated research program to understand the effective functional strategy of natural light-harvesting molecular antennae in photosynthetic organisms. This work tackles the problem of the structural optimization of light-harvesting antennae of variable size. In vivo, this size is controlled by light intensity during growth, thus implying more sophisticated optimization strategies, since larger antenna size demands finer structural tuning. Earlier modeling experiments showed that the aggregation of the antenna pigments, apart from being itself a universal structural factor optimizing the performance of light-harvesting antenna with any (!) spatial lattice, maintains its functioning provided that the degree of aggregation varies: the larger the unit building block, the higher the efficiency of the whole structure. It means that altering the degree of pigment aggregation in response to the antenna size is biologically expedient. In the case of the oligomeric chlorosomal antenna of green bacteria, the strategy of optimizing the variable antenna structure in response to the illumination intensity was demonstrated to take place in vivo and ensure high antenna efficiency regardless of its size, thus allowing bacteria to survive in a broad range of light intensities.

Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function.


III. MATHEMATICAL MODELS IN BIOLOGY

New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen.

MALDI-TOF MS and N-terminal amino acid sequencing allowed us to identify several fragments of the C-terminal peptide of Influenza A hemagglutinin (HA) containing transmembrane domains (TMD). These fragments were detected in the organic phase of chloroform-methanol extracts from bromelain-treated virus particles. Heterogeneous fatty acylation of the C-terminus was revealed. Tritium bombardment technique might open an opportunity for 3D structural investigation of the HA TMD in situ.

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.

Low gadolinium concentrations induce rapid gigaseal formation and cell adhesion to glass and plastic (polystyrene) substrates in the slime mutant of Neurospora crassa. Cellular adhesion is independent of an integrin-mediated mechanism, because pretreatment with the oligopeptide ARG-GLY-ASP-SER (RGDS) did not inhibit it, and there was no spatial correlation between integrin and adhesions. In contrast, concanavalin A and β-galactosidase both inhibit adhesion, suggesting that adhesion is mediated by sugar moeities at the cell surface. The adhesion sites are motile in the plasma membrane, as shown by the movement of polystyrene microspheres on the cell surface. In addition to an integrin-based adhesive system, which has already been characterized in walled hyphal cells, hyphae have evolved at least two different plasma membrane-based adhesion mechanisms. The relatively non-specific sugar-mediated adhesion caused by gadolinium may be part of the mechanism of gigaseal formation in other cells. In the absence of sugar-mediated adhesion, gadolinium increases the magnitude of the gigaseal in giant unilamellar liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, with or without the negatively charged phosphatidylserine. Thus, gigaseal formation involves at least two different mechanisms.

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wild-type strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm-1 region were measured for films of the intact and in situ trypsin-degraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm-1), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference ('wet' minus 'dry') spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.

Within the past 4 years, poliomyelitis outbreaks associated with circulating vaccine-derived polioviruses (cVDPVs) have occurred in Hispaniola (2000–01), the Philippines (2001), and Madagascar (2001–02). Retrospective studies have also detected the circulation of endemic cVDPV in Egypt (1988–93) and the likely localized spread of oral poliovirus vaccine (OPV)-derived virus in Belarus (1965–66). Gaps in OPV coverage and the previous eradication of the corresponding serotype of indigenous wild poliovirus were the critical risk factors for all cVDPV outbreaks. The cVDPV outbreaks were stopped by mass immunization campaigns using OPV. To increase sensitivity for detecting vaccine-derived polioviruses (VDPVs), in 2001 the Global Polio Laboratory Network implemented additional testing requirements for all poliovirus isolates under investigation. This approach quickly led to the recognition of the Philippines and Madagascar cVDPV outbreaks, but of no other current outbreaks. The potential risk of cVDPV emergence has increased dramatically in recent years as wild poliovirus circulation has ceased in most of the world. The risk appears highest for the type 2 OPV strain because of its greater tendency to spread to contacts. The emergence of cVDPVs underscores the critical importance of eliminating the last pockets of wild poliovirus circulation, maintaining universally high levels of polio vaccine coverage, stopping OPV use as soon as it is safely possible to do so, and continuing sensitive poliovirus surveillance into the foreseeable future. Particular attention must be given to areas where the risks for wild poliovirus circulation have been highest, and where the highest rates of polio vaccine coverage must be maintained to suppress cVDPV emergence.


IV. MOLECULAR VIROLOGY

RNA–protein interactions are fundamental for different aspects of molecular biology such as gene expression, assembly of biomolecular complexes or macromolecular transport. The 3a movement protein (MP) of a plant virus, Cucumber mosaic virus (CMV), forms ribonucleoprotein (RNP) complexes with viral RNA, capable of trafficking from cell-to-cell throughout the infected plant only in the presence of the CMV capsid protein (CP). However, deletion of the C-terminal 33 amino acid residues of the CMV MP (in the mutant designated 3aΔC33 MP) resulted in CP-independent cell-to-cell movement. The biological differences in the behaviour of CMV wild type (wt) 3a MP and 3aΔC33 MP could have been a consequence of differences in the RNA-binding properties of the two MPs detected previously using biochemical assays on ensembles of molecules. To investigate the physical mechanisms of MP–RNA interactions at a single molecule level, we applied atomic force microscopy to measure for the first time unbinding forces between these individual binding partners. Minimal unbinding forces determined for individual interaction of the CMV RNA molecule with the CMV wt or truncated MPs were estimated to be ~45 pN and ~90 pN, respectively, suggesting that the distinct differences in the strength of MP–RNA interactions for the wt MP and truncated MP are attributable to the molecular binding mechanism. We also demonstrated that molecules of both CMV 3a MP and 3aΔC33 MP were capable of self-interaction with minimal unbinding forces of ~50 pN and ~70 pN, respectively, providing a physical basis for the cooperative mechanism of the RNA binding. The significance of intermolecular force measurements for understanding the structural and functional aspects of viral RNP formation and trafficking is discussed.

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.

Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.

This review considers the results of probing the structure of ribonucleoprotein particles of helical plant viruses by tritium planigraphy (TP). This method works by exposing macromolecular targets to a beam of tritium atoms and analyzing the tritium label distribution along the macromolecule length. The TP data combined with theoretical predictions made it possible to propose a structural model of the coat protein for the virions of potato viruses X (the type representative of potexviruses) and A (a potyvirus), which eluded X-ray diffraction analysis so far. TP revealed fine structural differences between the wild-type tobacco mosaic virus (strain U1) and its temperature-sensitive mutant with an altered coat protein and host specificity. The possibil-ities of using TP for studying the RNA–protein interactions in helical virus particles are discussed.

The capsid protein (CP) of Cucumber mosaic virus (CMV) is required for cell-to-cell movement, mediated by the 3a movement protein (MP). Deletion of the C-terminal 33 amino acids of the CMV 3a MP (in the mutant designated 3a C33 MP) resulted in CP-independent cell-to-cell movement, but not long-distance movement. RNA-binding studies done in vitro using isolated bacterially expressed MP showed that the 3a C33 MP bound RNA more strongly, with fewer regions sensitive to RNase and formed cooperatively bound complexes at lower ratios of protein : RNA than the wild-type (wt) 3a MP. Analysis of the architecture of the complexes by atomic force microscopy showed that the wt 3a MP formed a single type of complex with RNA, resembling beads on a string. By contrast, the 3a C33 MP formed several types of complexes, including complexes with virtually no MP bound or thicker layers of MP bound to the RNA. Assays showed that protein–RNA complexes containing high levels of either MP inhibited the infectivity and in vitro translatability of viral RNAs. The 3a C33 MP inhibited these processes at lower ratios of protein : RNA than the wt 3a MP, consistent with its stronger binding properties. The apparent contradiction between these inhibition data and the CP-independent cell-to-cell movement of CMV expressing the 3a C33 MP is discussed.

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 520C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 μM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four α-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.


In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such “late” apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed.

Strong RNA silencing was induced in plants transformed with an amplicon consisting of full-length cDNA of potato leafroll virus (PLRV) expressing green fluorescent protein (GFP), as shown by low levels of PLRV-GFP accumulation, lack of symptoms and accumulation of amplicon-specific short interfering RNAs (siRNAs). Inoculation of these plants with various viruses known to encode silencing suppressor proteins induced a striking synergistic effect leading to the enhanced accumulation of PLRV-GFP, suggesting that it had escaped from silencing. However, PLRV-GFP escape also occurred following inoculation with viruses that do not encode known silencing suppressors and treatment of silenced plants with biotic or abiotic stress agents. We propose that viruses can evade host RNA-silencing defences by a previously unrecognized mechanism that may be associated with a host response to some types of abiotic stress such as heat shock.

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the “triple gene block” (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus), movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBp1 to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.

Previously, it has been shown that tobacco mosaic virus (TMV) U1 and crucifer-infecting TMV contain a 75 nt internal ribosome entry site (IRES) upstream of movement protein (MP) gene (IRESU1MP,75 and IRESCRMP,75, respectively). A movement-deficient TMV mutant, KK6, has been constructed previously [Lehto, K., Grantham, G. L. & Dawson, W. O. (1990). Virology 174, 145–157] by insertion of the second coat protein subgenomic promoter (CP SGP-2) upstream of the MP gene, in addition to the natural CP SGP-1. Here, the authors compare the efficiency of movement function expression by KK6 and a derivative, K86, obtained by insertion of IRESCRMP,75 between the CP SGP-2 and MP genes resulting in restoration of IRESCRMP,75 function in the 5'-untranslated sequence of the I2 subgenomic RNA of K86. The data indicate that the efficiency of K86 movement was largely restored by this insertion, which was apparently due to the translation-enhancing ability of IRESCRMP,75.

V. STRUCTURE, EXPRESSION AND EVOLUTION OF GENOM

Integrase (IN) is responsible for one of the key stages in the replication cycle of human immunodeficiency virus type 1, namely, integration of a DNA copy of the viral RNA into the infected cell genome. IN recognizes the nucleotide sequences located at the ends of the U3 and U5 regions of long terminal repeats (LTRs) of the viral DNA and sequentially catalyzes the 3'-end processing and strand transfer reactions. Analogs of U5 regions containing non-nucleoside insertions have been used to study the interaction between IN and viral DNA. Substrate modification has been demonstrated to have almost no effect on the rate of DNA binding by IN. However, the removal of heterocyclic bases from positions 5 and 6 of the substrate molecule and from position 3 of the processed strand almost completely inhibits IN enzymatic activity, which indicates the importance of these bases for the formation of an active enzyme–substrate complex. By contrast, modification of the third base of the nonprocessed strand stimulates 3'-processing. Since the base removal disturbs the complementary and stacking interactions in DNA, these results indicate that double-helix destabilization near the cleaved bond promotes 3'-end processing.

The early embryonic development of Nematoda proceeds by three ways, which strictly correspond to three phylogenetic lineages. Under the first way the endodermal precursor is localized in the posterior blastomere at the two-cells stage (such a determination is the peculiarity of all the Chromadoria, including Secernentea and Caenorhabditis elegans). Under the second way the endodermal precursor is localized in the anterior blastomere of the egg. This feature is very unusual for Metazoa, but it is the only way of entoderm determination in all the Dorylaimia orders (Mononchida, Mermithida, Trichinellida, Dioctophymida, Dorylaimida). The third way described for the sea Enoplida is characterized with variable location of blastomers and changeable localization of endodermal precursor before eight-cells stage. It is still unknown of these three variants was typical the most recent common ancestor of present Nematoda. D.A. Voronov (2001) produced argument in favour of variable cleavage as primitive one for Nematoda. This opinion is rejected because of the similarity in development between sea Enoplida and C. elegans. Both of them share such features as low-cell gastrula and neurula, identical phylotypic lima bean stage of embryogenesis, identity of some geometrical figures 4 or 8 blastomers, isolating of the endodermal precursor at the eight-cells stage, the lack in development of any plesiomorphous features, which are widely distributed outside Nematoda (under the variable cleavage of Enoplida there are no such locations of blastomers, which are typical for spiral or radial cleavage, there are no embryonic leaves as well). One can see the homology of separate cells at adult Enoplida and Rhabditia. Cell lineage of Triplonchida as far as it is described at Tobrilus gracilis doesn't exclude the hypothesis on their origin from the cleavage similar to one of present Dorylaimia with localization of the endodermal precursor in the anterior blastomere. In view of all the considerations mentioned above one should interpret variable cleavage of Enoplida as derivation from invariant cleavage.

Fragments of the nuclear and mitochondrial genes for the large-subunit rRNA were compared for Trichoplax sp. and T. adhaerens. High similarity was observed for their sequences, suggesting that different Trichoplax isolates belong to one species.

We have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant. The human and mouse pannexin-1 mRNAs are ubiquitously, although disproportionately, expressed in normal tissues. Human PANX2 is a brain-specific gene; its mouse orthologue, Panx2, is also expressed in certain cell types in developing brain. In silico evaluation of Panx3 expression predicts gene expression in osteoblasts and synovial fibroblasts. The apparent conservation of pannexins between species merits further investigation.

Dynamics of alterations of cell surface topography during TNF-induced apoptosis of HeLa cells was examined by phase-contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4-6 h of incubation but much earlier, after 1.5-3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1-2 h. This phenomenon was called "marginal blebbing". It took place before the cytochrome c release from mitochondria to cytosol. Marginal blebbing was inhibited by drugs that depolymerized actin microfilaments (cytochalasin, latrunculin) or decreased Rho-kinase-dependent contractility of actin-myosin cortex (H7, HA-1077, Y27632). A pancaspase inhibitor, zVAD-fmk, completely prevented marginal and general blebbing, and TNF-induced apoptosis. DEVD-fmk, a specific inhibitor of caspase-3, inhibited both marginal and general blebbing but not cell rounding and death. Thus, marginal blebbing is an early microfilament-dependent apoptotic event. It is suggested that it is initiated by minimal activation of caspase-3 and the following local Rho-kinase-dependent stimulation of actin-myosin cortex contractility. Localization of marginal blebs at the active edge may be associated with special organization of cortex in that zone.

Yang C.H., Murti A., Baker S.J., Frangou-Lazaridis M., Vartapetian A.B., Murti K.G. Pfeffer L.M.
Experimental Cell Research 298 (2004) 197-206.