I. biology of cell and cell organelles.

Activity of Different Classes of Neurons of the Motor Cortex during Postural Corrections.

Beloozerova I.N., Sirota M.G., Swadlow H.A., Orlovsky G.N., Popova L.B., Deliagina T.G.

The Journal of Neuroscience 23 (2003) 7844-7853.

The dorsal side-up body orientation in quadrupeds is maintained by a postural system that is driven by sensory feedback signals. The spinal cord, brainstem, and cerebellum play essential roles in postural control, whereas the role of the forebrain is unclear. In the present study we investigated whether the motor cortex is involved in maintenance of the dorsal side-up body orientation. We recorded activity of neurons in the motor cortex in awake rabbits while animals maintained balance on a platform periodically tilting in the frontal plane. The tilts evoked postural corrections, i.e., extension of the limbs on the side moving down and flexion on the opposite side. Because of these limb movements, rabbits maintained body orientation close to the dorsal side up. Four classes of efferent neurons were studied: descending corticofugal neurons of layer V (CF5s), those of layer VI (CF6s), corticocortical neurons with ipsilateral projection (CCIs), and those with contralateral projection (CCCs). One class of inhibitory interneurons [suspected inhibitory neurons (SINs)] was also investigated. CF5 neurons and SINs were strongly active during postural corrections. In most of these neurons, a clear-cut modulation of discharge in the rhythm of tilting was observed. This finding suggests that the motor cortex is involved in postural control. In contrast to CF5 neurons, other classes of efferent neurons (CCI, CCC, CF6) were much less active during postural corrections. This suggests that corticocortical interactions, both within a hemisphere (mediated by CCIs) and between hemispheres (mediated by CCCs), as well as corticothalamic interactions via CF6 neurons are not essential for motor coordination during postural corrections.

Postural Control in the Rabbit Maintaining Balance on the Tilting Platform.

Beloozerova I.N., Zelenin P.V., Popova L.B., Orlovsky G.N., Grillner S., Deliagina T.G.

J. Neurophysiol. 90 (2003) 3783-3793.

A deviation from the dorsal-side-up body posture in quadrupeds activates the mechanisms for postural corrections. Operation of these mechanisms was studied in the rabbit maintaining balance on a platform periodically tilted in the frontal plane. First, we characterized the kinematics and electromyographic (EMG) patterns of postural responses to tilts. It was found that a reaction to tilt includes an extension of the limbs on the side moving down and flexion on the opposite side. These limb movements are primarily due to a modulation of the activity of extensor muscles. Second, it was found that rabbits can effectively maintain the dorsal-side-up body posture when complex postural stimuli are applied, i.e., asynchronous tilts of the platforms supporting the anterior and posterior parts of the body. These data suggest that the nervous mechanisms controlling positions of these parts of the body can operate independently of each other. Third, we found that normally the somatosensory input plays a predominant role for the generation of postural responses. However, when the postural response appears insufficient to maintain balance, the vestibular input contributes considerably to activation of postural mechanisms. We also found that an asymmetry in the tonic vestibular input, caused by galvanic stimulation of the labyrinths, can affect the stabilized body orientation while the magnitude of postural responses to tilts remains unchanged. Fourth, we found that the mechanisms for postural corrections respond only to tilts that exceed a certain (threshold) value.

Locomotion of CV-1 cytoplasts with or without a centrosome.

Burakov A.V.

Tsitologiia 45 (2003) 132-142.

The movement of cultured cells along the substratum is a convenient model for studying cell movement in the organism, occurring during embryogenesis, angiogenesis, metastasis, wound closure, etc. The moving cells must control their pseudopodial activity along the perimeter, regulate the attachment and reattachment to the substratum, and pull their body following pseudopodium during their movement along the substratum. As proven by numerous investigations, these processes directly depend on the actomyosin system of cells. The role of microtubules as components of cytoskeleton in cell locomotion still remains obscure. The role of microtubules in cell movement is commonly investigated using microtubule-destructive drugs. Therefore in the final results the accessory drug effect on, for example, signal cascades cannot be excluded. Another mode of action on the microtubule dynamics is centrosome removal from the cells, which is easily realized by their removal together with the nucleus. It has been shown that in cytoplasts of centrosome containing fibroblasts, dynamic instability of microtubules remains. Unlike, in non-centriolar cytoplasts tread milling is observed. Some literature evidence suggests that cytoplasts of cultured cells move along the substratum not worse that intact cells do. In this study cytoplasts with and without centrosome were obtained and identified, and parameters of movement along the substratum (speed, direction) were registered for both these two populations of cytoplasts, and for control intact cells and cells involved in the experiment. The model of experimental wound of monolayer was used, because it guaranteed cell synchronization in respect to movement direction and speed. Centrosome-containing CV-1 cytoplasts displayed radial microtubule array, and centrosome-lacking cytoplasts exhibited chaotic distribution of microtubules, which is characteristic of microtubule tread milling. In addition, both kinds of cytoplasts appeared to move along the substratum much slower than the whole cells. No difference was found is speed and keeping direction between centriolar and non-centriolar cytoplasts.

Membrane Permeability Changes at Early Stages of Influenza hemagglutinin-Mediated Fusion.

Frolov V.A., Dunina-Barkovskaya A.Y., Samsonov A.V., Zimmerberg J.

Biophysical Journal 85 (2003) 1725-1733.

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.

Shape bistability of a membrane neck: A toggle switch to control vesicle content release.

Frolov V.A., Lizunov V.A., Dunina-Barkovskaya A.Ya., Samsonov A.V., Zimmerberg J.

Proc. Natl. Acad. Sci. USA 100 (2003) 8698–8703.

Shape dynamics and permeability of a membrane neck connecting a vesicle and plasma membrane are considered. The neck is modeled by a lipid membrane tubule extended between two parallel axisymmetric rings. Within a range of lengths, defined by system geometry and mechanical properties of the membrane, the tubule has two stable shapes: catenoidal microtubule and cylindrical nanotubule. The permeabilities of these two shapes, measured as ionic conductivity of the tubule interior, differ by up to four orders of magnitude. Near the critical length the transitions between the shapes occur within less than a millisecond. Theoretical estimates show that the shape switching is controlled by a single parameter, the tubule length. Thus the tubule connection can operate as a conductivity microswitch, toggling the release of vesicle content in such cellular processes as “kiss-and-run” exocytosis. In support of this notion, bistable behavior of membrane connections between vesicles and the cell plasma membrane in macrophages is demonstrated.

α-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts.

Hinz B., Dugina V., Ballestrem C., Wehrle-Haller B., Chaponnier C.

Molecular Biology of Cell 14 (2003) 2508-2519.

Cultured myofibroblasts are characterized by stress fibers, containing -smooth muscle actin (α-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of α-SMA–negative fibroblasts. We have investigated the role of α-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of α-SMA–negative fibroblasts. Then, myofibroblasts incorporate α-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When α-SMA expression is induced in 5 d culture by TGF or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with α-SMA fusion peptide (SMA-FP), which inhibits α-SMA–mediated contractile activity, reduces their adhesion to the level of α-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and 3 integrin-containing FAs; β₅ integrin distribution is not affected. FRAP of 3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high α-SMA–mediated contractile activity of myofibroblast stress fibers.

Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation.

Ivanov P.A., Chudinova E.M., Nadezhdina E.S.

Experimental Cell Research 290 (2003) 227-233.

Stress granules are RNP-containing particles arising in the cytoplasm in response to environmental stress. They are dynamic structures assembling and disassembling in the cytoplasm very rapidly. We have studied whether the cytoskeleton is involved in the formation of stress granules. Stress granules were induced in CV-1 cells by sodium arsenate treatment and visualized by immunofluorescent staining with antibodies either to the p170 subunit of eIF3 or to poly(A)-binding protein. Treatment with sodium arsenate for 30–120 min led to assembling of stress granules in a majority of CV-1 cells. Disruption of MT array with nocodazole treatment abolished arsenate-induced formation of stress granules. A similar effect was induced by the microtubule-depolymerizing drug vinblastine, though the influence of the microtubule-stabilizing drug paclitaxel was opposite. Nocodazole treatment did not prevent arsenate-induced phosphorylation of the eIF-2 factor, essential for stress granule formation, suggesting that the presence of intact MT array is required for granule assembly. Unexpectedly, treatment of cells with the actin filament-disrupting drug latrunculin B slightly enhanced stress granule formation. We propose that stress granule formation is microtubule-dependent process and likely is facilitated by the motor protein-driven movement of individual stress granule components (e.g., mRNP) along microtubules.

A new human gene KCNRG encoding potassium channel regulating protein is a cancer suppressor gene candidate located in 13q14.3.

Ivanov D.V., Tyazhelova T.V., Lemonnier L., Kononenko N., Pestova A.A., Nikitin E.A, Prevarskaya N., Skryma R., Panchin Y.V., Yankovsky N.K., Baranova A.V.

FEBS Letters 539 (2003) 156-160.

We report the primary characterization of a new gene KCNRG mapped at chromosome band 13q14.3. This gene includes three exons and has two alternatively spliced isoforms that are expressed in normal tissues and in some tumor cell lines. Protein KCNRG has high homology to tetramerization domain of voltage-gated K⁺ channels. Using the patch-clamp technique we determined that KCNRG suppresses K⁺ channel activity in human prostate cell line LNCaP. It is known that selective blockers of K⁺ channels suppress lymphocyte and LNCaP cell line proliferation. We suggest that KCNRG is a candidate for a B-cell chronic lymphocytic leukemia and prostate cancer tumor suppressor gene.

Effects of troponin on thermal unfolding of actin-bound tropomyosin.

Kremneva E.V., Nikolaeva O.P., Gusev N.B., Levitsky D.I.

Biochemistry (Moscow) 68 (2003) 802-809.

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8⁰C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48⁰C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70⁰C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100⁰C. However, addition of TnI, and especially of TnT, to the F-actin-Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem.

Lazareva E.M., Polyakov V.Y., Chentsov Yu.S., Smirnova E.A.

Cell Biol. Int. 27 (2003) 633-646.

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of α-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of α-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of α-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.

Fusion, fragmentation, and fission of mitochondria.

Polyakov V.Y., Soukhomlinova M.Y., Fais D.

Biochemistry (Moscow) 68 (2003) 838-849.

Individual mitochondria which form the chondriom of eucaryotic cells are highly dynamic systems capable of fusion and fragmentation. These two processes do not exclude one another and can occur concurrently. However, fragmentation and fusion of mitochondria regularly alternate in the cell cycle of some unicellular and multicellular organisms. Mitochondrial shapes are also described which are interpreted as intermediates of their "equational" division, or fission. Unlike the fragmentation, the division of mitochondria, especially synchronous division, is also accompanied by segregation of mitochondrial genomes and production of specific "dumbbell-shaped" intermediates. This review considers molecular components and possible mechanisms of fusion, fragmentation, and fission of mitochondria, and the biological significance of these processes is discussed.

Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain.

Potekhina E.S., Zinovkina L.A., Nadezhdina E.S.

Biochemistry (Moscow) 68 (2003) 188-195.

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro.

Serazev T.V., Nadezhdina E.S., Shanina N.A., Leshchiner A.D., Kalinina N.O., Morozov S.Yu.

Molecular Biology (Moscow) 37 (2003) 919-925.

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.

Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells.

Uzbekov R., Timirbulatova E., Watrin E., Cubizolles F., Ogereau D., Gulak P., Legagneux V., Polyakov V.J., Le Guellec K., Kireev I.

J. Cell. Sci. 116 (2003) 1667-1678.

Cell cycle dynamics and localization of condensins - multiprotein complexes involved in late stages of mitotic chromosome condensation - were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to beta-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF, fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

The step-wise assembly of a functional nucleolus in preim-plantation mouse embryos involves the cajal (coiled) body.

Zatsepina O., Baly C., Chebrout M., Debey P.

Dev. Biol. 253 (2003) 66-83.

After fertilization, ribosomal RNA synthesis is silenced during a period which depends on the species. Data concerning the reassembly of a functional nucleolus remain scarce. We have examined by immunocytochemistry, Western blots, and BrUTP microinjection the dynamics of major nucleolar proteins during the first cycles of mouse embryogenesis, in relation to rDNA transcription sites and coilin, a marker of Cajal bodies. We show that: (1) the reinitiation of rDNA transcription occurs at the two-cell stage, 44-45 h after hCG injection (hphCG), at the surface of the nucleolar precursor bodies (NPBs), where the RNA polymerase I (pol I) transcription complex is recruited 4-5 h before; (2) the NPBs are not equal in their ability to support recruitment of pol I and rDNA transcription; (3) maternally inherited fibrillarin undergoes a dynamic redistribution during the second cell stage, together with coilin, leading to the assembly of the Cajal body around 40 hphCG; and (4) the pol I complex is first recruited to the Cajal body before reaching its rDNA template. We also find that fibrillarin and B23 are both directly assembled around NPBs prior to ongoing pre-rRNA synthesis. Altogether, our results reveal a role of the Cajal bodies in the building of a functional nucleolus.

II. Bioenergetics and photosynthesis.

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca²⁺.

Bodrova M.E., Brailovskaya I.V., Efron G.I., Starkov A.A., Mokhova E.N.

Biochemistry (Moscow) 68 (2003) 391-398.

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential Δψ across the inner mitochondrial membrane caused by 10 μM myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the "classic" state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of "classic pore" opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive Δψ decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped Δψ decrease induced by 5 μM myristic acid and 5 μM Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the Δψ decrease, and the following addition of EGTA produced only a weak (if any) Δψ increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the Δψ decrease. These data show that the cyclosporin A-sensitive decrease in Δψ by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive Δψ decrease results from a conversion of Δψ to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.

The beginnings of research on biophysics of photosynthesis and initial contributions made by Russian scientists to its development.

Borisov A.

Photosynthesis Research 76 (2003) 413-426.

In contrast to the classical sciences, biophysics is difficult to define. For example, Roderick Clayton suggested that biophysics requires 'solid grounding in physics, chemistry and mathematics together with enough biology and biochemistry' [Clayton RK (1988) Photosynth.Res 19: 207-224]. One may see from the proceedings of the recent biophysical congresses that their materials and ideas in a very wide sense are biological, including global geographic and ecological problems. To be recognized as biophysical, either physico-chemical methods or at least some mathematical and computer programs are usually involved in such work. One exception is the biophysics of photosynthesis, which deals with fundamental photophysical processes: the absorption of solar radiation by chlorophylls (Chls) and accessory pigments. The subsequent intermolecular transfer of singlet electronic excitation results in a primary energy conversion manifested as pairs of opposite electric charges separated in the pigment-protein complexes called reaction centers [see Clayton RK (2002) Photosynth Res 73: 63-71]. I review the initial, basic contributions in this field, and the most important accomplishments of Russian scientists in the 20th century.

Mechanisms of excitation trapping in reaction centers of purple bacteria.

Borisov A.Y.

Biochemistry (Moscow) 68 (2003) 152-161.

The contradiction between two groups of experimental data, which fails to be resolved within the framework of the widely accepted model of excitation migration and trapping (at least in case of purple bacteria), is discussed in the introduction to this review. Three directions of studies intended to resolve this conflict are reviewed in the three further sections: II. Exciton models; III. Water-polarization (water-latch) mechanism of excitation trapping; IV. Quantum-mechanical models. The maximum efficiency of these models in resolving the contradiction mentioned above was assessed. The advantages and disadvantages of the mechanisms described in sections II, III, and IV are discussed in the last section of this review. It is concluded that none of these mechanisms taken alone is able to solve this problem. Therefore, the fundamental problem of the primary excitation conversion in reaction centers remains unsolved and requires additional experimental research.

The revision of the model of primary energy conversion in purple bacteria.

Borisov A.Y., Sidorin Y.M.

Bioelectrochemistry 59 (2003) 113-119.

A simulation method is suggested which enables one to check whether a model for excitation energy exchange in an ensemble of dye molecules fits available experimental data. In particular, this method may deal with photosynthetic units (PSUs) in which excitation migration in antenna chlorophylls and their substantial trapping in reaction centers (RCs) take place. Its application to the purple bacteria has proved that the model, which was generally accepted during the last 20-30 years, is in contradiction with recent experimental facts and thus requires modernization. Two physical mechanisms are discussed: femtosecond polarization of mobile hydrogen atoms near the reaction center special pair ("water latch"), and the presence of excitons delocalized over several core-bacteriochlorophylls (BChls). Our considerations give evidence that neither of these mechanisms alone can resolve the conflict, but their cumulative action appears to be sufficient. Unfortunately, these mechanisms were as yet only partially addressed experimentally.

Two-level heterogenous energy migration. Modeling and application to purple bacteria.

Borisov A.Yu., Vasil'kov S.L.

Biofizika 48 (2003) 40-48.

The dynamics of migration of electronic excitations and the efficiency of their trapping in two-dimensional ensembles of molecules were analyzed. Molecules were characterized using the following parameters: the width of long-wavelength bands, the values of extinction and rate constant of deactivation of electronic excitations, critical distances of migration close to those of dye molecules, in particular, bacteriochlorophyll a and purple bacteria. A comparative analysis of two-dimensional models of energy migration made it possible to chose a model with an optimum light-harvesting on traps from the largest numbers of light-absorbing molecules. It was shown that in ensembles of molecules having different spectral characteristics (spectral shifts between the short- and long-wavelength fractions of the molecules are hear 800 cm-1) the efficiency of excitation trapping is approximately 90 and 80% for the number of light-harvesting molecules per one trap 210 and 580, respectively.

Low Dielectric Permittivity of Water at the Membrane Interface: Effect on the Energy Coupling Mechanism in Biological Membranes.

Cherepanov D.A, Feniouk B.A., Junge W., Mulkidjanian A.Y.

Biophysical Journal 85 (2003) 1307-1316.

Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (pH^B) or between two membrane surfaces (pH^S). To scrutinize whether pH^S can deviate from pH^B, we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, Phys. Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5–1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2–0.3 eV) than for monovalent cations (0.1–0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH^S of 6.0; the value of pH^S was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.

Kindling fluorescent proteins for precise in vivo photolabeling.

Chudakov D.M., Belousov V.V., Zaraisky A.G., Novoselov V.V., Staroverov D.B., Zorov D.B., Lukyanov S., Lukyanov K.A.

Nature Biotechnology 21 (2003) 191-194.

Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

Two Generations of Insulinotropic Imidazoline Compounds.

Efendic S., Efanov A.M., Berggren P.-O., Zaitsev S.V.

Diabetes 51 (2002) S448-S454.

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K⁺ channels as well as voltage-gated K⁺ channels, which led to membrane depolarization, an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca²⁺]_i but in the presence of high ATP and Ca²⁺concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K⁺ channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis.

Functional Analysis of the Na⁺/H⁺ Antiporter Encoding Genes of the Cyanobacterium Synechocystis PCC 6803.

Elanskaya I.V., Karandashova I.V., Bogachev A.V., Hagemann M.

Biochemistry (Moscow) 67 (2002) 432-440.

The role of putative Na⁺/H⁺ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na⁺/H⁺ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na⁺/H⁺ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na⁺/H⁺ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na⁺/H⁺ antiporters was studied. No induction of any Na⁺/H⁺ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na⁺/H⁺ antiporter encoding genes showed induction. These results might indicate that some of Na⁺/H⁺ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.

RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase.

Gasman S., Kalaidzidis Y., Zerial M.

Nature Cell Biology 5 (2003) 195–204.

Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.

A function for novel uncoupling proteins: antioxidant defense of mitochondrial matrix by translocating fatty acid peroxides from the inner to the outer membrane leaflet.

Goglia F., Skulachev V.P.

The FASEB Journal 17 (2003) 1585-1591.

It is hypothesized that mitochondrial uncoupling proteins operate as carriers of fatty acid peroxide anions. This is assumed to result in electrophoretic extrusion of such anions from the inner to the outer leaflet of the inner mitochondrial membrane, being driven by membrane potential (mitochondrial interior negative). In this way, the inner leaflet is ridded of fatty acid peroxides that otherwise can form very aggressive oxidants damaging mitochondrial DNA, aconitase, and other mitochondrial matrix-localized components of vital importance. The steady-state concentration the fatty acid peroxides is known to be low. This explains why UCP2, 3, 4, and 5 are present in small amounts usually insufficient to make a large contribution to the H⁺ conductance of the mitochondrial membrane.

Ion transport coupled to terminal oxidase functioning in the extremely alkaliphilic halotolerant bacterium Thioalkalivibrio.

Grischuk Y.V., Muntyan M.S., Popova I.V., Sorokin D.Y.

Biochemistry (Moscow) 68 (2003) 385-390.

Proton transport in the terminal part of the respiratory chain in the extremely alkaliphilic halotolerant bacterial strain Thioalkalivibrio versutus was studied under near-optimum growth conditions (pH 9.0-9.5). Under these conditions, bacterial cells generated electric potential with the negative charge being inside the cells. When only the terminal part of the respiratory chain functioned, it was found that: 1) unlike other bacteria known, this bacterium did not acidify the medium in the presence of K⁺ and valinomycin; 2) in the presence of an uncoupler, CCCP, but in the absence of valinomycin, reversible alkalinization of the medium occurred as a result of proton influx into the cells. Cyanide prevented this alkalinization. The difference spectra indicate that cell membranes contained cytochromes c and (b+o), some of which reacted with CO. The respiratory activity of membranes in the terminal part of the respiratory chain was optimal at pH 9.5 and specifically depended on sodium ions (C_1/2 = 10 mM). The data suggest the presence of a Na⁺-pump in the terminal part of the respiratory chain of the studied strain which can pump Na⁺ out of the cells.

Occupancy of two primary chloride-binding sites in Natronobacterium pharaonis halorhodopsin is a necessary condition for active anion transport.

Kalaidzidis I.V., Kalaidzidis Y.L.

Biochemistry (Moscow) 68 (2003) 354-358.

The existence of two primary chloride-binding sites was found on the basis of the study of halorhodopsin spectra at different chloride concentrations. SVD analysis of the spectra revealed two chloride-dependent components at low chloride concentration (0.1-10 mM). Global fitting of SVD components found K(D) values of 0.47 and 5.2 mM with unitity Hill coefficients. The second K_D coincides with the apparent K_D of the photovoltage response of halorhodopsin.

Cyclosporin A-sensitive cytochrome c release and activation of external pathway of NADH oxidation in liver mitochondria due to pore opening by acidification of phosphate-containing incubation medium.

Knorre D.A., Dedukhova V.I., Vyssokikh M.Y., Mokhova E.N.

Biosci. Rep. 23 (2003) 67-75.

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.

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells.

Kohler M., Zaitsev S.V., Zaitseva I.I., Leibiger B., Leibiger I.B., Turunen M., Kapelioukh I.L., Bakkman L., Appelskog I.B., Boutet De Monvel J., Imreh G., Berggren P.O.

Diabetes 52 (2003) 2943-2950.

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 μmol/l), showing that this method worked in insulin-producing cells.

Detection and functional role of local gradients of H⁺-ions on the intracellular mitochondrial membrane with covalently linked pH-probe.

Kozlova M.V., Gramadskii K.B., Solodovnikova I.M., Krasinskaya I.P., Vinogradov A.V., Yaguzhinskii L.S.

Biofizika 48 (2003) 443-452.

The study is devoted to the registration of local H⁺ gradients on the inner membrane of mitochondria under conditions of H⁺ pump functioning were recorded. By using a covalently linked pH probe (fluorescein isothiocyanate), a local increase in the activity of hydrogen ions on the outer face of the inner mitochondrial membrane in the presence of the respiration substrate at increased permeability of the membrane for K⁺ was registered. It was also found that the buffer capacity of medium affects the respiration rate of completely uncoupled mitochondria; a change in respiration rate strictly correlates with changes in local H⁺ gradients on the mitochondrial membrane. It was concluded that local gradients of H⁺ activity can control the rate of functioning of H⁺ pumps. It was shown that, under certain conditions, the system of H⁺ pumps incorporated into succinate oxidase of mitochondria functions as a nonliner system.

The structure of BtuB with bound colicin E3 R-domain implies a translocon.

Kurisu G., Zakharov S.D., Zhalnina M.V., Bano S., Eroukova V.Y., Rokitskaya T.I., Antonenko Y.N., Wiener M.C., Cramer W.A.

Nature Structural Biology 10 (2003) 948-954.

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-Å coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 Å is reported. Binding of R135 to the BtuB extracellular surface (G° = -12 kcal mol^-1) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135–BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin α.

Markova OV, Evstafieva AG, Mansurova SE, Moussine SS, Palamarchuk LA, Pereverzev MO, Vartapetian AB, Skulachev VP.

Biochimica et Biophysica Acta 1557 (2003) 109-117.

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin α (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin α (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H₂O₂, catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.

Background Subtraction in Experimental Data Arrays Illustrated by the Example of Raman Spectra and Fluorescent Gel Electrophoresis Patterns.

Mikhailyuk I.K., Razjivin A.P.

Instruments and Experimental Techniques 46 (2003) 765-769.

.A method for background signal subtraction from one-dimensional experimental data arrays (optical spectra, chromatograms) and two-dimensional arrays (electrophoregrams) is based on the difference in the geometric properties (radii of curvature) of the curves of the useful and background signals. To demonstrate the efficiency of this method, it is applied to the Raman spectrum of chymotrypsin and a fluorescent gel electrophoregram of hepatitis B DNA fragments stained with ethydium bromide.

Decomposing Spectra into Bands Using the Initial Spectrum and a Set of Its Derivatives.

Mikhailyuk I.K., Razjivin A.P.

Biofizika 48 (2003) 383-388.

A method for the decomposition of optical spectra into bands was proposed, which is based on simultaneous approximation of the initial spectrum and its derivatives. The bands of the standard (gaussian) form were used in the decomposition procedure. A method for the selection of the optimal smoothing filter was described. The efficiency of the proposed method was demonstrated on model and experimental absorption spectra. It was shown that this method makes it possible to determine the number of bands and its parameters more exactly than the standard approach based on the analysis of one derivative of one power.

Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light.

Mulkidjanian A.Y., Cherepanov D.A., Galperin M.Y.

BMC Evol. Biol. 28 (2003) 12.

A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world"). However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth. RESULTS: As condensation of sugar phosphates and nitrogenous bases is thermodynamically unfavorable, these compounds, if ever formed, should have undergone rapid hydrolysis. Thus, formation of oligonucleotide-like structures could have happened only if and when these structures had some selective advantage over simpler compounds. It is well known that nitrogenous bases are powerful quenchers of UV quanta and effectively protect the pentose-phosphate backbones of RNA and DNA from UV cleavage. To check if such a protection could play a role in abiogenic evolution on the primordial Earth (in the absence of the UV-protecting ozone layer), we simulated, by using Monte Carlo approach, the formation of the first oligonucleotides under continuous UV illumination. The simulations confirmed that UV irradiation could have worked as a selective factor leading to a relative enrichment of the system in longer sugar-phosphate polymers carrying nitrogenous bases as UV-protectors. Partial funneling of the UV energy into the condensation reactions could provide a further boost for the oligomerization. CONCLUSION: These results suggest that accumulation of the first polynucleotides could be explained by their abiogenic selection as the most UV-resistant biopolymers.

Exciton modeling of energy-transfer dynamics in the lhcii complex of higher plants: a redfield theory approach.

Novoderezhkin V., Salverda J.M., van Amerongen H., van Grondelle R.

J. Phys. Chem. B 107 (2003) 1893-1912.

We propose an exciton model for the peripheral plant light-harvesting complex LHCII that allows us to explain the absorption (OD) and linear dichroism (LD) spectra, the superradiance (SR), the pump-probe transient absorption (TA), the three-pulse photon echo peak shift (3PEPS), and transient grating (TG) kinetics at different excitation wavelengths. To calculate the nonlinear response we used the Liouville equation for the density matrix, expanded up to the third order with respect to the external field, with the Redfield relaxation superoperator in the exciton eigenstate basis. We found a few configurations of the antenna, with specific chlorophyll (Chl) a/b identities, orientations, and site energies, that allowed a simultaneous fit of these data while taking into account the excitonic interactions, the static disorder, and weak exciton-phonon coupling which induced dephasing and relaxation (energy transfer) between the exciton states. The spectral density of the exciton-phonon coupling adjusted from the fit allowed us to determine the time scales and pathways of energy transfer in LHCII. We find that the intraband (Chl bàChl b and Chl aàChl a) energy-transfer dynamics includes sub-picosecond (250-600 fs) exciton relaxation within dimeric or, in the Chl a band, more complicated clusters, sub-picosecond (600-800 fs) hopping between spatially separated clusters (in the a band), and “slow” (picosecond) migration between localized states. The interband (Chl bàChl a) transfer is characterized by the presence of very fast channels, the fastest taking only 120 fs, which connect both localized and dimeric b states with the a band. The overall relaxation/migration dynamics can be directly viewed by means of the time-dependent density matrix in the exciton and site representation. The latter representation allows us to visualize the time-dependent degree of delocalization. While the individual exciton states can be delocalized over 2-2.5 molecules in the Chl a region, thermal mixing results in a coherence size of 1.4-1.8 for the steady-state wave packet at room temperature. Altogether, we conclude that the experimentally determined dynamic and static properties of LHCII can be simulated very well on the basis of the proposed excitonic model.

intra- and interband transfers in the b800-850 antenna of Rhodospirillum molishianum: redfield theory modeling of polarized pump-probe kinetics.

Novoderezhkin V., Wendling M., van Grondelle R.

J. Phys. Chem. B 107 (2003) 11534-11548.

We use an exciton model for the B800-B850 LH2 light-harvesting antenna of Rhodospirillum molischianum to explain the absorption, excitation-wavelength-dependent pump-probe kinetics, and induced absorption anisotropy at 77 K reported previously (Wendling, M.; van Mourik, F.; van Stokkum, I. H. M.; Salverda, J. M.; Michel, H.; van Grondelle, R. Low-intensity pump-probe measurements on the B800 band of Rhodospirillum molischianum. Biophys. J. 2003, 84, 440). The nonlinear response was calculated using the density matrix equation, expanded up to the third-order with respect to the external field, with the Redfield relaxation operator in the exciton basis. The model allowed us to obtain a simultaneous and quantitative fit of the data while taking into account the excitonic interactions, the static disorder, and phonon-induced relaxation of populations and coherences in the one-exciton manifold (including a nonsecular coherence transfer and population-coherence coupling). The spectral density of the exciton-phonon coupling adjusted from the fit allowed us to determine the time scales and pathways of energy transfer. The 800 nm band consists of exciton states of the outer ring (B800 states) together with the upper Davydov component of the inner ring (B850* states). The 850 nm band contains the lower Davydov states of the inner ring (B850 states). The excitation dynamics includes migration around the outer ring with 1-3 ps time constant (the B800 à B800 transfer), 1 ps transfer to the inner ring (B800 à B850 transfer), and 600-800 fs transfer to higher states of the inner ring (B800 à B850* transfer). The dynamics of B850* states is characterized by very fast 100-200 fs intraband (B850* à B850*) equilibration, 60-200 fs interband (B850* à B850) relaxation, and a slower (>250 fs) back (B850* à B800) transfer. The relative contribution of the B800 à B850* à B850 pathway is comparable (approximately equal) to the contribution of the direct B800 à B850 transfer. Both B800 à B800 and B800 à B850* transfers are faster for the blue side of the 800 nm band, giving rise to slower kinetics when the excitation wavelength is tuned from 788 to 800 nm. From 800 to 809 nm kinetics become faster due to the increasing contribution of the directly excited B850* states and due to better coupling (faster relaxation) to higher states of the B850 band. The anisotropy decay in the 800 nm region exhibits a fast component (300-500 fs), reflecting the decay of the one-exciton coherences that is followed by slow picosecond depolarization due to B800 à B800 migration. Both these processes can be directly viewed by means of the density matrix in the exciton and site representation.

Cytochrome c, an ideal antioxidant.

Pereverzev M.O., Vygodina T.V., Konstantinov A.A., Skulachev V.P.

Biochem. Soc. Trans. 31 (2003) 1312-1315.

Generation of Δψ (membrane potential) by cytochrome oxidase proteoliposomes oxidizing superoxide-reduced cytochrome c has been demonstrated. XO+HX (xanthine oxidase and hypoxanthine) were used to produce superoxide. It was found that the generation of Δψ is completely abolished by cyanide (an uncoupler) or by superoxide dismutase, and is enhanced by nigericin. Addition of ascorbate after XO+HX causes a further increase in Δψ. On the other hand, XO+HX added after ascorbate do not affect Δψ, indicating that superoxide does not have measurable protonophorous activity. The half-maximal cytochrome c concentration for Δψ generation supported by XO+HX was found to be approx. 1 microM. These data and the results of some other researchers can be rationalized as follows: (1) O₂ accepts an electron to form superoxide; (2) cytochrome c oxidizes superoxide back to O₂; (3) an electron removed from the reduced cytochrome c is transferred to O₂ by cytochrome oxidase in a manner that generates Δμ(H⁺) (transmembrane difference in electrochemical H⁺ potential). Thus cytochrome c mediates a process of superoxide removal, resulting in regeneration of O₂ and utilization of the electron involved previously in the O₂ reduction. It is important that cytochrome c is not damaged during the antioxidant reaction, in contrast with many other antioxidants.

Optical Absorption and Circular Dichroism Spectra of Light-Harvesting Complexes from Rhodopseudomonas acidophila as Calculated from X-Ray Diffraction Data.

Pishchal’nikov R.Yu., Razjivin A.P.

Biofizika 48 (2003) 207-212.

Absorption and circular dichroism spectra of two forms of the peripheral light-harvesting complex from photosynthetic purple bacteria Rps. acidophila were calculated. Calculations were carried out on the basis of exciton theory for circular aggregates of bacteriochlorophyll molecules and X-ray data for these forms of the complex. It was shown that theoretical spectra fit well experimental ones for the same values of excitation energy, homogeneous and inhomogeneous boarding and bandwidth for all bacteriochlorophyll molecules of complexes. To fit CD spectra one need to suppose additional small orientation changes of transition moment Q_y relative to its orientation according X-ray data.

Tandem gramicidin channels cross-linked by streptavidin.

Rokitskaya T.I., Kotova E.A., Antonenko Y.N.

J. Gen. Physiol. 121 (2003) 463-476.

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.

Cytochrome c decelerates channel kinetics of negatively charged gramicidin due to electrostatic interaction.

Rokitskaya T.I., Kotova E.A., Antonenko Y.N.

Biochemical and Biophysical Research Communications 302 (2003) 865-868.

The effect of cytochrome c on the kinetic properties of ion channels formed by O-pyromellitylgramicidin (OPg), the negatively charged analogue of gramicidin A (gA), in bilayer lipid membranes was studied by the method of sensitized photoinactivation. The addition of cytochrome c to both sides of the membrane caused substantial deceleration of the photoinactivation kinetics of OPg channels which expose three negative charges to the aqueous phase at both sides of the membrane. By contrast, the gA photoinactivation kinetics was unaltered by the addition of cytochrome c. Based on the sensitivity of the observed effect to the ionic strength of the bathing solution, the cytochrome c-induced deceleration of the OPg photoinactivation kinetics reflecting the increase in the OPg channel lifetime was ascribed to electrostatic interaction of positive charges of cytochrome c with negative charges of OPg that resulted in channel clustering. Formation of clusters of OPg channels was previously inferred to explain the polylysine effect on the OPg channel kinetics. The decelerating effect of cytochrome c on OPg channels was observed only at a high number of OPg channels in the membrane, thus suggesting that the interaction between cytochrome c and the charged transmembrane protein requires sufficiently high negative charge density on the surface of the membrane.

Energy Transfer in Light-Harvesting Complexes LHCII and CP29 of Spinach Studied with Three Pulse Echo Peak Shift and Transient Grating.

Salverda J.M., Vengris M., Krueger B.P., Scholes G.D., Czarnoleski A.R., Novoderezhkin V., van Amerongen H., van Grondelle R.

Biophysical Journal 84 (2003) 450-465.

Three pulse echo peak shift and transient grating (TG) measurements on the plant light-harvesting complexes LHCII and CP29 are reported. The LHCII complex is by far the most abundant light-harvesting complex in higher plants and fulfills several important physiological functions such as light-harvesting and photoprotection. Our study is focused on the light-harvesting function of LHCII and the very similar CP29 complex and reveals hitherto unresolved excitation energy transfer processes. All measurements were performed at room temperature using detergent isolated complexes from spinach leaves. Both complexes were excited in their Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. Exponential fits to the TG and three pulse echo peak shift decay curves were used to estimate the timescales of the observed energy transfer processes. At 650 nm, the TG decay can be described with time constants of 130 fs and 2.2 ps for CP29, and 300 fs and 2.8 ps for LHCII. At 670 nm, the TG shows decay components of 230 fs and 6 ps for LHCII, and 300 fs and 5 ps for CP29. These time constants correspond to well-known energy transfer processes, from Chl b to Chl a for the 650 nm TG and from blue (670 nm) Chl a to red (680 nm) Chl a for the 670 nm TG. The peak shift decay times are entirely different. At 650 nm we find times of 150 fs and 0.5–1 ps for LHCII, and 360 fs and 3 ps for CP29, which we can associate mainly with Chl b Chl b energy transfer. At 670 nm we find times of 140 fs and 3 ps for LHCII, and 3 ps for CP29, which we can associate with fast (only in LHCII) and slow transfer between relatively blue Chls a or Chl a states. From the occurrence of both fast Chl b Chl b and fast Chl b Chl a transfer in CP29, we conclude that at least two mixed binding sites are present in this complex. A detailed comparison of our observed rates with exciton calculations on both CP29 and LHCII provides us with more insight in the location of these mixed sites. Most importantly, for CP29, we find that a Chl b pair must be present in some, but not all, complexes, on sites A3 and B3. For LHCII, the observed rates can best be understood if the same pair, A3 and B3, is involved in both fast Chl b Chl b and fast Chl a Chl a transfer. Hence, it is likely that mixed sites also occur in the native LHCII complex. Such flexibility in chlorophyll binding would agree with the general flexibility in aggregation form and xanthophyll binding of the LHCII complex and could be of use for optimizing the role of LHCII under specific circumstances, for example under high-light conditions. Our study is the first to provide spectroscopic evidence for mixed binding sites, as well as the first to show their existence in native complexes.

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes.

Semenov A.Y., Mamedov M.D., Chamorovsky S.K.

FEBS Letters 553 (2003) 223-228.

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c₆), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c₆ through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.

Structural Proton Diffusion along Lipid Bilayers.

Serowy S., Saparov S.M., Antonenko Y.N., Kozlovsky W., Hagen V., Pohl P.

Biophysical Journal 84 (2003) 1031-1037.

For H⁺ transport between protein pumps, lateral diffusion along membrane surfaces represents the most efficient pathway. Along lipid bilayers, we measured a diffusion coefficient of 5.8 x 10^-5 cm² s^-1. It is too large to be accounted for by vehicle diffusion, considering proton transport by acid carriers. Such a speed of migration is accomplished only by the Grotthuss mechanism involving the chemical exchange of hydrogen nuclei between hydrogen-bonded water molecules on the membrane surface, and the subsequent reorganization of the hydrogen-bonded network. Reconstitution of H⁺-binding sites on the membrane surface decreased the velocity of H⁺ diffusion. In the absence of immobile buffers, structural (Grotthuss) diffusion occurred over a distance of 100 µm as shown by microelectrode aided measurements of the spatial proton distribution in the immediate membrane vicinity and spatially resolved fluorescence measurements of interfacial pH. The efficiency of the anomalously fast lateral diffusion decreased gradually with an increase in mobile buffer concentration suggesting that structural diffusion is physiologically important for distances of 10 nm.

Coupling of nuclear wavepacket motion and charge separation in bacterial reaction centers.

Shuvalov V.A., Yakovlev A.G.

FEBS Letters 540 (2003) 26-34.

The mechanism of the charge separation and stabilization of separated charges was studied using the femtosecond absorption spectroscopy. It was found that nuclear wavepacket motions on potential energy surface of the excited state of the primary electron donor P* leads to a coherent formation of the charge separated states P⁺B_A^-, P⁺H_A^- and P⁺H_B^- (where B_A, H_B and H_A are the primary and secondary electron acceptors, respectively) in native, pheophytin-modified and mutant reaction centers (RCs) of Rhodobacter sphaeroides R-26 and in Chloroflexus aurantiacus RCs. The processes were studied by measurements of coherent oscillations in kinetics at 890 and 935 nm (the stimulated emission bands of P*), at 800 nm (the absorption band of B_A) and at 1020 nm (the absorption band of B_A^-) as well as at 760 nm (the absorption band of H_A) and at 750 nm (the absorption band of H_B). It was found that wavepacket motion on the 130-150 cm^-1 potential surface of P* is accompanied by approaches to the intercrossing region between P* and P⁺B_A^- surfaces at 120 and 380 fs delays emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A^-). In the presence of Tyr M210 (Rb. sphaeroides) or M195 (C. aurantiacus) the stabilization of P⁺B_A^- is observed within a few picosseconds in contrast to YM210W. At even earlier delay (approximately 40 fs) the emission at 895 nm and bleaching at 748 nm are observed in C. aurantiacus RCs showing the wavepacket approach to the intercrossing between the P* and P⁺H_B^- surfaces at that time. The 32 cm^-1 rotation mode of HOH was found to modulate the electron transfer rate probably due to including of this molecule in polar chain connecting P_B and B_A and participating in the charge separation. The mechanism of the charge separation and stabilization of separated charges is discussed in terms of the role of nuclear motions, of polar groups connecting P and acceptors and of proton of OH group of TyrM210.

Glucose Metabolites Inhibit Protein Phosphatases and Directly Promote Insulin Exocytosis in Pancreatic ß-Cells

Sjöholm A., Lehtihet M., Efanov A.M., Zaitsev S.V., Berggren P.-O., Honkanen R.E.

Endocrinology 143 (2003) 4592-4598.

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event. Glucose is the cardinal physiological stimulator of insulin secretion from the pancreatic ß-cell, but the mechanisms involved in glucose sensing are not fully understood. Specific ser/thr protein phosphatase (PPase) inactivation by okadaic acid promotes Ca²⁺ entry and insulin exocytosis in the ß-cell. We now show that glycolytic and Krebs cycle intermediates, whose concentrations increase upon glucose stimulation, not only dose dependently inhibit ser/thr PPase enzymatic activities, but also directly promote insulin exocytosis from permeabilized ß-cells. Thus, fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate, and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis, nonadditive to that of okadaic acid, at micromolar Ca²⁺ concentrations. In contrast, the effect of GTP is potentiated by okadaic acid, suggesting that the action of GTP does not require PPase inactivation. We conclude that specific glucose metabolites and GTP inhibit ß-cell PPase activities and directly stimulate Ca²⁺-independent insulin exocytosis. The glucose metabolites, but not GTP, seem to require PPase inactivation for their stimulatory effect on exocytosis. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by metabolic intermediates, may be a novel regulatory mechanism linking glucose sensing to insulin exocytosis in the ß-cell.

A Risky Job: In search of noncanonical pathways. Selected topics in the history of biochemistry: personal recollections VII. G. Semenza and A.J. Turner (Eds.)

Skulachev V.P.

Comprehensive Biochemistry 42 (2003) 319-410.

When my old friend Giorgio Semenza asked me to write this chapter, I immediately accepted his kind invitation. I did this not only because it is great honor to write for this prominent Comprehensive Biochemistry collection but also because I am now 67, an age to turn back and see what was already done and to avoid mistakes in the future. With age, mistakes become more and more expensive...

Programmed death phenomena at various levels of development of the living systems.

Skulachev V.P.

In: Formal descriptions of developing systems. J. Nation et al. (eds). Kluwer Publishers, Netherlands, pp. 61-86 (2003).

A concept will be presented assuming that any complex biological system, from intracellular organelles (mitochondria) to multicellular organisms is equipped with a program of self-elimination. This suicide program is actuated when the system in question proved to be unwanted for a system occupying a higher position in biological hierarchy. The concept called “Samurai law of biology” (“It is better to die than to be wrong”) will be illustrated considering defence of organelles, cells, organs and organisms against damaging effects of reactive oxygen species (ROS). (1) In bacteria, DNA oxidation by ROS initiates (a) induction of synthesis of reparation enzymes, (b) arrest of the cell divisions and (c) autolysin activation resulting in the programmed death (PD), depending on dose of ROS and duration of their action. In mitochondria, ROS open the permeability transition pore, initiating in this way PD of mitochondria (mitoptosis), which purifies mitochondrial population from the ROS-overproducing organelles. In yeast, H₂O₂ induces some proteins, including caspase, causing PD. Inhibition of the protein synthesis prevents these effects. Also in yeast, high levels of a pheromone proved to cause ROS formation resulting in PD. In human HeLa cells, we found that tumor necrosis factor (TNF) initiates ROS formation and then PD (apoptosis). At supracellular level, the PD signal is transmitted from the TNF-treated to intact cells and such a transmission is arrested by catalase, indicating that H₂O₂ serves as an intercellular PD messenger. Conversion of a tadpole to a frog was shown to be mediated by thyroxine causing induction of a NO synthase in the tadpole tail cells. This results in strong increase in the H₂O₂ level due to inhibition by NO of catalase and gluthatione peroxidase. Moreover, NO causes antimycin A-like inhibition of mitochondrial respiratory chain, which strongly stimulates ROS production. Due to massive apoptosis, the tail disappears (organoptosis). It is suggested that ROS mediate aging which is considered as programmed death of organism (phenoptosis). The “Samurai law” is regarded as a mechanism preventing the great destructive potential of environment, as well as of the living systems per se, from being realized. It helps organisms to maintain intact their genomes that were developed during billion years of evolution but can be destroyed during several generations by a single mutation in one of thousands genome-composing genes.

H₂O₂ Sensors of lungs and blood vessels and their role in the antioxidant defense of the body.

Skulachev V.P.

In: Free Radicals, Nitric Oxide and Inflammation. Molecular, Biochemical and Clinical Aspects. A. Tomasi, et al. eds. IOS Press Amsterdam, pp. 232-236 (2003).

This paper considers the composition and function of sensory systems monitoring H₂O₂ level by the lung neuroepithelial and carotid body. These systems are localized in the plasma membrane of cells of the corresponding organs and are composed of (i) O₂^.--generating NADPH-oxidase and (ii) an H₂O₂-activated K⁺ channel. This complex structure of the H₂O₂ sensors is probably due to their function in the antioxidant defense. By means of these sensors, an increase in the H₂O₂ level in lung or blood results in a decrease in lung ventilation and constriction of blood vessels. This action lowers the O₂ flux to the tissues and, hence, intracellular [O₂]. The [O₂] decrease, in turn, inhibits intracellular generation of reactive oxygen species. The possible roles of such systems under normal conditions (e.g., the effect of O₂^.- in air) and in some pathologies (e.g., pneumonia) is discussed.

Alternative functions of mitochondria.

Skulachev V.P.

In: Free Radicals, Nitric Oxide and Inflammation. Molecular, Biochemical and Clinical Aspects. A. Tomasi, et al. eds. IOS Press Amsterdam, pp. 1-7 (2003).

Mitochondria are known to be multifuctional intracellular organelles. They carry out (i) energy conservation in forms of protonic potential (Dm_H+) and ATP, (ii) thermoregulatory energy dissipation as heat, (iii) production of useful substances, (iv) decomposition of harmful substances, and (v) regulation of intracellular processes. It is suggested that mitochondria are equipped by a mechanism of self-elimination (“mitoptosis”) responsible for purification of mitochondrial population from unwanted organelles (e.g., ROS-overproducing mitochondria). Massive mitoptosis is assumed to induce apoptosis due to release of the cell death proteins normally hidden in the intermembrane space of mitochondria. In this way tissues are purified from ROS-overproducing and other unwanted cells.

Aging and the programmed death phenomena.

Skulachev V.P.

In: Topics in Current Cenetics, Vol. 3 (T. Nystrom and H.D. Osiewacz, Eds.) Model systems in ageing. Springer-Verlag Berlin Heidelberg, pp. 191-238 (2003).

Biochemical mechanisms of the programmed death phenomena are considered at levels of unicellular organisms, mitochondria, cells, groups of cells and organs. Some examples of programmed death of multicellular organisms are also discussed. A concept is developed that aging, being inherent in the great majority of eukaryotic organisms, is programmed, operating as a mechanism accelerating evolution. It is assumed that genetic changes which are too small to be a subject of natural selection in strong young individuals, become essential with age, and, hence, can be selected, at older (but still reproductive) ages when the organism is weakened by aging. A general scheme of the programmed aging is described. It postulates involvement of a kind of biological clock, aging or juvenile hormones, reactive oxygen species, telomeres, some specific intracellular aging-mediating proteins (p53, p66Shc), oxidation of mitochondrial DNA and proteins, nuclear DNA demethylation, etc. It is concluded that the programmed aging concept should be regarded as an alternative to the traditional point of view regarding aging as being due to inevitable damage to long-term operating complex living systems.

Substitutions for glutamate 101 in subunit II of cytochrome c oxidase from Rhodobacter sphaeroides result in blocking the proton-conducting K-channel.

Tomson F.L., Morgan J.E., Gu G., Barquera B., Vygodina T.V., Gennis R.B.

Biochemistry 42 (2003) 1711-1717.

Two functional input pathways for protons have been characterized in the heme-copper oxidases: the D-channel and the K-channel. These two proton-conducting channels have different functional roles and have been defined both by X-ray crystallography and by the characterization of site-directed mutants. Whereas the entrance of the D-channel is well-defined as D132(I) (subunit I; Rhodobacter sphaeroides numbering), the entrance of the K-channel has not been clearly defined. Previous mutagenesis studies of the cytochrome bo(3) quinol oxidase from Escherichia coli implicated an almost fully conserved glutamic acid residue within subunit II as a likely candidate for the entrance of the K-channel. The current work examines the properties of mutants of this conserved glutamate in the oxidase from R. sphaeroides (E101(II)I,A,C,Q,D,N,H) and residues in the immediate vicinity of E101(II). It is shown that virtually any substitution for E101(II), including E101(II)D, strongly reduces oxidase turnover (to 8-29%). Furthermore, the low steady-state activity correlates with an inhibition of the rate of reduction of heme a₃ prior to the reaction with O₂. These are phenotypes expected of K-channel mutants. It is concluded that the predominant entry point for protons going into the K-channel of cytochrome oxidase is the surface-exposed glutamic acid E101(II) in subunit II.

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue.

Tonshin A.A., Saprunova V.B., Solodovnikova I.M., Bakeeva L.E., Yaguzhinsky L.S.

Biochemistry (Moscow) 68 (2003) 875-881.

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000 g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700 g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000 g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue--small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements--"mitochondrion in mitochondrion". The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.

EPR study of light-induced regulation of photosynthetic electron transport in Synechocystis sp. strain PCC 6803.

Trubitsin B.V., Mamedov M.D., Vitukhnovskaya L.A., Semenov A.Yu., Tikhonov A.N.

FEBS Letters 544 (2003) 15-20.

The kinetics of the light-induced redox changes of the photosystem 1 (PS 1) primary donor P₇₀₀ in whole cells of the cyanobacteria Synechocystis sp. PCC 6803 were studied by the electron paramagnetic resonance method. It was shown that the linear photosynthetic electron transport in cyanobacteria was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from PS 1 to NADP⁺ due to activation of the Calvin cycle reactions and (ii) retardation of electron flow between two photosystems governed by a transmembrane proton gradient. In addition to the linear photosynthetic electron transport, cyanobacteria were capable of maintaining alternative pathways involving cyclic electron transfer around PS 1 and respiratory chains.

The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis.

Vyssokikh M.Y., Brdiczka D.

Acta Biochim Pol. 50 (2003) 389-404.

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and 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 creatine kinase octamer. However, when, for example, 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.

Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect.

Vyssokikh M.Y., Zorova L., Zorov D., Heimlich G., Jurgensmeier J.J., Brdiczka D.

Mol. Biol. Rep. 29 (2002) 93-96.

The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-AC released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-AC. (III) The Bax-AC effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-AC effect was as well suppressed by hexokinase phosphorylating glucose.

Femtosecond nuclear oscillations under charge separation in reaction centers of photosynthesis.

Yakovlev A.G., Shkuropatov A.Y., Shuvalov V.A.

Biochemistry (Moscow) 68 (2003) 541-550.

Results are presented of a study of primary processes of formation of the charge separated states P⁺B_A^- and P⁺H_A^- (where P is the primary electron donor, B_A and H_A the primary and secondary electron acceptors) in native and pheophytin-modified reaction centers (RCs) of Rhodobacter sphaeroides R-26 by methods of femtosecond spectroscopy of absorption changes at low temperature. Coherent oscillations were studied in the kinetics at 935 nm (P* stimulated emission band), at 1020 nm (B_A^- absorption band), and at 760 nm (H_A absorption band). It was found that when the wavepacket created under femtosecond light excitation approaches the intersection between P* and P⁺B_A^- potential surfaces at 120- and 380-fsec delays, the formation of two electron states emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A^-) takes place. At the later time the wavepacket motion has a frequency of 32 cm^-1 and is accompanied by electron transfer from P* to B_A in pheophytin-modified and native RCs and further to H_A in native RCs. It was shown that electron transfer processes monitored by the 1020-nm absorption band development as well as by bleaching of 760-nm absorption band have the enhanced 32 cm^-1 mode in the Fourier transform spectra.

Electron transfer in deuterated reaction centers of Rhodobacter sphaeroides at 90 K according to femtosecond spectroscopy data.

Yakovlev A.G., Shuvalov V.A.

Biochemistry (Moscow) 68 (2003) 603-610.

The primary act of charge separation was studied in P⁺B_A^- and P⁺H_A^- states (P, primary electron donor; B_A and H_A, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of B_A^- (1020 nm) and of absorption band of H_A (760 nm). It was found that in native RCs kept in heavy water (D₂O) buffer the isotopic decreasing of basic oscillation frequency 32 cm^-1 and its overtones takes place by the same factor approximately 1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H₂O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm^-1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor P_B and primary electron acceptor B_A. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to B_A. The results allow for tracing of the possible pathway of electron transfer from P* to B_A along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(P_B)-N-C-N(His M200)-HOH-O = B_A. We assume that the role of 32- cm^-1 modulation in electron transfer along this chain consist of a fixation of electron density at B_A^- during a reversible electron transfer, when populations of P* and P⁺B_A^- states are approximately equal.

Mechanism of Charge Separation and Stabilization of Separated Charges in Reaction Centers of Chloroflexus aurantiacus and of YM210W(L) Mutants of Rhodobacter sphaeroides Excited by 20 fs Pulses at 90 K.

Yakovlev A.G., Vasilieva L.G., Shkuropatov A.Ya., Bolgarina T.I., Shkuropatova V.A., Shuvalov V.A.

J. Phys. Chem. A 107 (2003) 8330-8338.

The nuclear wave packet formed by 20 fs excitation on the P* potential energy surface in native and mutant (YM210W and YM210L) reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides and in Chloroflexus (C.) aurantiacus RCs was found to be reversibly transferred to the P⁺B_A^- surface at 120, 380, etc. fs delays (monitored by measurements of B_A^- absorption at 1020-1028 nm). The YM210W(L) mutant RCs show the most simple pattern of femtosecond oscillations with a period of 230 fs in stimulated emission from P* and with the initial amplitude comparable to that in plant pheophytin a (Pheo)-modified Rb. sphaeroides R-26 RCs. Similar reversible oscillations are observed in the 1020 nm band of the mutants, the initial amplitude of which is smaller by a factor of ~10 with respect to Pheo-modified Rb. sphaeroides R-26 RCs. In contrast to native and Pheo-modified Rb. sphaeroides R-26 RCs, irreversible quasi-exponential stabilization of P⁺B_A^- is considerably suppressed in the mutant RCs in the picosecond time domain. The water rotational mode with a frequency of 32 cm^-1 and its overtones, described earlier (Yakovlev; et al. Biochemistry 2002, 41, 2667-2674), are decreased in the YM210W(L) mutants and strongly suppressed in dry films of the mutant RCs. In the dry film of both YM210W and YM210L RCs neither reversible nor irreversible P⁺B_A^- formation monitored at 1020 nm is observed despite the preservation of fs oscillations with a frequency of 144 cm^-1 in the 935 nm kinetics of stimulated emission from P*. Furthermore, the 1020 nm band is not formed inside of P*. In C. aurantiacus RCs, containing leucine instead of tyrosine at the M208 position, the P* decay is slowed to ~5 ps at 90 K (1.5 ps in Rb. sphaeroides RCs) and characterized by fs oscillations with the amplitude comparable to that measured in native Rb. sphaeroides R-26 RCs. The B_A^- absorption band development at 1028 nm is observed at 90 K with fs oscillations similar to those described for native Rb. sphaeroides R-26 RCs at 293 K but with the amplitude being smaller by a factor of ~6. The kinetics of absorbance changes in the 1028 nm band in C. aurantiacus RCs includes the stabilization of P⁺B_A^- within ~5 ps with subsequent decay due to electron transfer to H_A within ~1 ps. The mechanisms of the electron-transfer pathway between P* and B_A and of the stabilization of the state P⁺B_A^- in bacterial RCs are discussed.

III. Mathematical models in biology.

IV. Molecular virology.

Microarray analysis of evolution of RNA viruses: Evidence of circulation of virulent highly divergent vaccine-derived polioviruses.

Cherkasova E., Laassri M., Chizhikov V., Korotkova E., Dragunsky E., Agol V.I., Chumakov K.

Proc. Natl. Acad. Sci. USA 100 (2003) 9398-9403.

Two approaches based on hybridization of viral probes with oligonucleotide microarrays were developed for rapid analysis of genetic variations during microevolution of RNA viruses. Microarray analysis of viral recombination and microarray for resequencing and heterogeneity analysis were able to generate instant genetic maps of vaccine-derived polioviruses (VDPVs) and reveal the degree of their evolutionary divergence. Unlike conventional methods based on cDNA sequencing and restriction fragment length polymorphism, the microarray approaches are better suited for analysis of heterogeneous populations and mixtures of different strains. The microarray hybridization profile is very sensitive to the cumulative presence of small quantities of different mutations, including those that cannot be revealed by sequencing, making this approach useful for characterization of profiles of nucleotide sequence diversity in viral populations. By using these methods, we identified a type-3 VDPV isolated from a healthy person and missed by conventional methods of screening. The mutational profile of the polio strain was consistent with >1 yr of circulation in human population and was highly virulent in transgenic mice, confirming the ability of VDPV to persist in communities despite high levels of immunity. The proposed methods for fine genotyping of heterogeneous viral populations can also have utility for a variety of other applications in studies of genetic changes in viruses, bacteria, and genes of higher organisms.

Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.

Gorshkova E.N., Erokhina T.N., Stroganova T.A., Yelina N.E., Zamyatnin A.A. Jr, Kalinina N.O., Schiemann J., Solovyev A.G., Morozov S.Y.

Journal of General Virology 84 (2003) 985-994.

The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.

Dysfunctionality of a tobacco mosaic virus movement protein mutant mimicking threonine 104 phosphorylation.

Karger E.M., Frolova O.Y., Fedorova N.V., Baratova L.A., Ovchinnikova T.V., Susi P., Makinen K., Ronnstrand L., Dorokhov Y.L., Atabekov J.G.

Journal of General Virology 84 (2003) 727-732.

Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr₁₀₄ in TMV MP. The MP-specific PKs with apparent molecular masses of about 45-50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr₁₀₄ by neutral Ala or by negatively charged Asp. Mutation of Thr₁₀₄ to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr₁₀₄ phosphorylation.

Retrospective analysis of a local cessation of vaccination against poliomyelitis: a possible scenario for the future.

Korotkova E.A., Park R., Cherkasova E.A., Lipskaya G.Y., Chumakov K.M., Feldman E.V., Kew O.M., Agol V.I.

Journal of Virology 77 (2003) 12460-12465.

The global eradication of poliomyelitis will require substantial changes in immunization practices. One of the proposed scenarios includes cessation of vaccination with live oral poliovirus vaccine (OPV) and the creation of an OPV stockpile for emergency response in case of the reintroduction of poliovirus into circulation. We describe here a retrospective analysis of the cessation of OPV usage in a region of the Byelorussian Republic of the former Soviet Union in 1963 to 1966. During this period, a widespread circulation and evolution of independent lineages of vaccine-derived polioviruses took place in the region. Some of these lineages appeared to originate from OPV given to 40 children in the community during this period of essentially no vaccinations. The data demonstrate very high risks associated with both the local cessation of OPV vaccination and the proposed use of OPV to control a possible reemergence of poliovirus in the postvaccination period. The high transmissibility of OPV-derived viruses in nonimmune population, documented here, and the known existence of long-term OPV excretors should be also considered in assessing risks of the synchronized global cessation of OPV usage.

Triple gene block: modular design of a multifunctional machine for plant virus movement.

Morozov S.Y., Solovyev A.G.

Journal of General Virology 84 (2003) 1351-1366.

Many plant virus genera encode a 'triple gene block' (TGB), a specialized evolutionarily conserved gene module involved in the cell-to-cell and long-distance movement of viruses. The TGB-based transport system exploits the co-ordinated action of three polypeptides to deliver viral genomes to plasmodesmata and to accomplish virus entry into neighbouring cells. Although data obtained on both the TGB and well-studied single protein transport systems clearly demonstrate that plant viruses employ host cell pathways for intra- and intercellular trafficking of genomic nucleic acids and proteins, there is no integral picture of the details of molecular events during TGB-mediated virus movement. Undoubtedly, understanding the molecular basis of the concerted action of TGB-encoded proteins in transporting viral genomes from cell to cell should provide new insights into the general principles of movement protein function. This review describes the structure, phylogeny and expression of TGB proteins, their roles in virus cell-to-cell movement and potential influence on host antiviral defences.

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein.

Rafikova E.R., Kurganov B.I., Arutyunyan A.M., Kust S.V., Drachev V.A., Dobrov E.N.

Int. J. Biochem. Cell Biol. 35 (2003) 1452-1460.

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15 s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

Zinovkin R.A., Erokhina T.N., Lesemann D.E., Jelkmann W., Agranovsky A.A.

Journal of General Virology 84 (2003) 2265-2270.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His₆ was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly₅₈₈/Gly₅₈₉ bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

V. structure, expression and evolution

of Genom

Distinct contributions of TNF and LT cytokines to the develop-ment of dendritic cells in vitro and their recruitment in vivo.

Abe K., Yarovinsky F.O., Murakami T., Shakhov A.N., Tumanov A.V., Ito D., Drutskaya L.N., Pfeffer K., Kuprash D.V., Komschlies K.L., Nedospasov S.A.

Blood 1001 (2003) 1477-1483.

TNF/LT/LT (tumor necrosis factor/lymphotoxin-/lymphotoxin-) triple knockout (KO) mice show a significant reduction of dendritic cell (DC) number in the spleen, presumably due to defective recruitment and/or production. To distinguish between these possibilities, DCs were generated from bone marrow (BM) cultures prepared from wild-type (wt) and mutant mice in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield of CD11c+ major histocompatibility complex (MHC) class II+ DCs generated from TNF/LT/LT/ BM culture was significantly reduced compared with wt BM culture. In order to further dissect the individual pathways responsible for defective DC properties observed in TNF/LT/LT/ mice, the panel of TNF/LT ligand and receptor single KO mice were used. The production of DCs from BM culture was significantly reduced in TNF/ and TNF receptor (TNFR) p55/ mice, but normal in LT/, LT/, LTR/ mice. Recombinant TNF (rTNF) exogenously added to TNF/LT/LT/ BM cultures could reverse this defect, and blocking antibodies showed partial effect on BM cultures of wt mice. Conversely, numbers of mature DCs in spleen were significantly decreased in LT/, LT/, LTR/ mice, but not in TNF/ and TNFRp55/ mice. These results reveal 2 distinct contributions of TNF/LT cytokines. First, TNF acting through TNF receptor is involved in the development/maturation of DCs in BM progenitor cultures, but this function appears to be redundant in vivo. Second, the microenvironment in peripheral lymphoid organs associated with LT/LT-LTR signaling and chemokine production is critical for recruitment efficiency of DCs, and this pathway is indispensable.

On interrelation between genetic systematics and genomics.

Antonov A.S.

Zhurnal Obshcheii Biologii 64 (2003) 181-186.

Review papers describing recent achievements of genomics usually do not pay attention to direct interrelation between genomics and genosystematics (DNA-systematics). Genomics on general is based in complete DNA sequencing of genomes. Initial aim of genosystematics was the same. Absence of historical perspective in review papers devoted to genomics decreases its value. In case it is done deliberately it becomes the problem of scientific ethics. It is postulated that genomics is a natural stage of genosystematics (DNA-systematics) development. Russian scientists were among the founders of these branches of biology.

Atomic tritium as a surface nanoprobe in a structural investigation of molecular assemblies.

Badun G.A., Lukashina E.V., Batuk O.N., Ksenofontov A.L., Fedoseev V.M.

Materials Science and Engineering C 23 (2003) 797–802

Possibilities of atomic tritium application as surface nanoprobe for structural investigations of adsorption layers on the liquid–air interface have been demonstrated. Frozen aqueous solutions of a series of amino acids and their mixtures and one well-known surface-active substance (cetyltrimethylammonium bromide, CTAB) were exposed to bombardment by tritium atoms generated on hot tungsten wire in a special vacuum device. This procedure resulted in substitution of hydrogen atoms by radioactive tritium in the thin surface layer of investigated samples. Curves of radioactivity changes depending on bombardment time and solution concentration for applied compounds were obtained and analyzed.

DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins.

Borisova O.A., Romanenkov A.S., Metelev V.G., Kubareva E.A., Zubin E.M., Timchenko M.A., Oretskaia T.S.

Molecular Biology (Moscow) 37 (2003) 534-543.

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting.

Dmitriev S.E., Pisarev A.V., Rubtsova M.P., Dunaevsky Y.E., Shatsky I.N.

FEBS Letters 533 (2003) 99-104.

A method of analysis of translation initiation complexes by toeprinting has recently acquired a wide application to investigate molecular mechanisms of translation initiation in eukaryotes. So far, this very fruitful approach was used when researchers did not aim to discriminate between patterns of toeprints for 48S and 80S translation initiation complexes. Here, using cap-dependent and internal ribosomal entry site (IRES)-dependent mRNAs, we show that the toeprint patterns for 48S and 80S complexes are distinct whether the complexes are assembled in rabbit reticulocyte lysate or from fully purified individual components. This observation allowed us to demonstrate for the first time a delay in the conversion of the 48S complex into the 80S complex for β-globin and encephalomyocarditis virus (EMCV) RNAs, and to assess the potential of some 80S antibiotics to block polypeptide elongation. Besides, additional selection of the authentic initiation codon among three consecutive AUGs that follow the EMCV IRES was revealed at steps subsequent to the location of the initiation codon by the 40S ribosomal subunit.

Assembly of 48S Translation Initiation Complexes from Purified Components with mRNAs That Have Some Base Pairing within

Their 5' Untranslated Regions.

Dmitriev S.E., Terenin I.M., Dunaevsky Y.E., Merrick W.C., Shatsky I.N.

Mol. Cell Biol. 23 (2003) 8925-8933.

The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5' UTR of β-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5' UTRs (with the cumulative stability of the secondary structure within the entire 5' UTR < -13 kcal/mol) and not essential for β-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5' UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.

Minor secondary-structure variation in the 5'-untranslated region of the β-globin mRNA changes the concentration requirements for eIF2.

Dmitriev S.E., Terenin I.M., Rubtsova M.P., Shatskii I.N.

Molecular Biology (Moscow) 37 (2003) 494-503.

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure elements in the 5'-untranslated region (5'-UTR) of the β-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at its low concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF3B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the β-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the complex of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR.

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Dobrov E.N., Badun G.A., Lukashina E.V., Fedorova N.V., Ksenofontov A.L., Fedoseev V.M., Baratova L.A.

European Journal of Biochemistry 270 (2003) 3300-3308.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.

Effect of Antioxidant BHT on the Proteolytic Apparatus of Aging Coleoptiles of Wheat Seedlings Grown in Light.

Dunaevskii Ya.E., Aleksandrushkina N.I., Smirnova T.A., Kolomiitseva G.Ya., Vanyushin B.F., Belozersky M.A.

Russian Journal of Bioorganic Chemistry 29 (2003) 459-463.

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old \seedlings. Under the effect of BHT, the plants grown in the light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.

Apoptosis-related fragmentation, translocation, and properties of human prothymosin α.

Evstafieva A.G., Belov G.A., Rubtsov Y.P., Kalkum M., Joseph B., Chichkova N.V., Sukhacheva E.A., Bogdanov A.A., Pettersson R.F., Agol V.I., Vartapetian A.B.

Exp Cell Res. 284 (2003) 211-323.

Human prothymosin α is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin α fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin α at one major carboxy terminal (DDVD₉₉) and several suboptimal sites. Prothymosin α cleavage at two amino-terminal sites (AAVD₆ and NGRD₃₁) contributes significantly to the final pattern of prothymosin α fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D₉₉ disrupts the nuclear localization signal of prothymosin α, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin α monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin α in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin α and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

Fedoreyeva L.I., Aleksandrushkina N.I., Dunaevsky Y.E., Belozersky M.A., Vanyushin B.F.

Biochemistry (Moscow) 68 (2003) 464-469.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Ivanov S., Alekseev Y., Bertrand J.R., Malvy C., Gottikh M.B.

Nucleic Acids Research 31 (2003) 4256-4263.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.

Solid phase synthesis and chromatographic characteristics of nucleophilic agents conjugated with oligonucleotides containing 5'-terminal carboxyl group.

Kachalova A.V., Tashlitskii V.N., Stetsenko D.A., Romanova E.A., Gait M.J., Oretskaia T.S.

Russian Journal of Bioorganic Chemistry 29 (2003) 303-309.

Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC.

Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome.

Kiryanov G.I., Isaeva L.V., Kinzurashvili L.N., Zacharova M.G.

Biochemistry (Moscow) 68 (2003) 492-496.

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.

Isolation and Primary Structure of Trypsin from the King Crab Paralithodes camtschaticus.

Kislitsyn Yu.A., Rebrikov D.V., Dunaevskii Ya. E., Rudenskaya G.N.

Russian Journal of Bioorganic Chemistry 29 (2003) 242-248.

Trypsin from hepatopancreas of the crab Paralithodes camtschaticus was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. A cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the shrimp Penaeus vannamei appeared to be closest in primary structure to that of the king crab (65% identity).

Effects of various N-terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria.

Kovaleva I.E., Novikova L.A., Nazarov P.A., Grivennikov S.I., Luzikov V.N.

European Journal of Biochemistry 270 (2003) 222-229.

Topogenesis of cytochrome p450scc, a resident protein of the inner membrane of adrenocortical mitochondria, is still obscure. In particular, little is known about the cause of its tissue specificity. In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome p450scc as its native precursor (pCYP11A1) or versions in which its N-terminal addressing presequence had been replaced with those of yeast mitochondrial proteins: CoxIV(1-25) and Su9(1-112). We found the pCYP11A1 and CoxIV(1-25)-mCYP11A1 versions to be effectively imported into yeast mitochondria and subjected to proteolytic processing. However, only minor portions of the imported proteins were incorporated into mitochondrial membranes, whereas their bulk accumulated as aggregates insoluble in 1% Triton X-100. Along with previously published data, this suggests that a distinguishing feature of the import of the CYP11A1 precursors into yeast mitochondria is their easy translocation into the matrix where the foreign proteins mainly undergo proteolysis or aggregation. The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively converted into the catalytically active holoenzyme. Experiments with the Su9(1-112)-mCYP11A1 construct bearing a re-export signal revealed that, after translocation of the fused protein into the matrix and its processing, the Su9(67-112) segment ensures association of the mCYP11A1 body with the inner membrane, but proper folding of the latter does not take place. Thus it can be said that the most specific stage of CYP11A1 topogenesis in adrenocortical mitochondria is its confinement and folding in the inner mitochondrial membrane. In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism.

Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation.

Krylova O.O., Melik-Nubarov N.S., Badun G.A., Ksenofontov A.L., Menger F.M., Yaroslavov A.A.

Chemistry 9 (2003) Aug 18; 9(16): 3930-6.

It has recently been found that Pluronics (block copolymers of ethylene oxide, EO, and propylene oxide, PO) favor the permeability and accumulation of anthracycline antibiotics, for example doxorubicin (Dox), in tumor cells. In an effort to understand these results, the interaction of EO(2)/PO(32)/EO(2) (Pluronic L61) with unilamellar egg yolk vesicles (80-100 nm in diameter) was examined. A partition coefficient K_p = [Pl](membrane)/[Pl](wa-ter)=45 was determined. This corresponds to adsorption of about 20 polymer molecules to the surface of each vesicle in a 20 μM polymer solution. Despite this rather weak adsorption, Pluronic has a substantial effect upon the transmembrane permeation rate of Dox and upon the phospholipid flip-flop rate within the bilayers. Thus, the Dox permeation rate increases threefold and the flip-flop rate increases sixfold in 20 μM Pluronic. The two rates increase linearly with the amount of adsorbed polymer. The obvious ability of Pluronics to increase the mobility of membrane components may have important biomedical consequences.

Soluble tankyrase located in cytosol of human embryonic kidney cell line 293.

Kuimov A.N., Terekhov S.M.

Biochemistry (Moscow) 68 (2003) 260-268.

We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types.

Use of crosslinking for revealing the DNA phosphate groups forming specific contacts with the E. coli Fpg protein.

Kuznetsova S., Rykhlevskaya A., Taranenko M., Sidorkina O., Oretskaya T., Laval J.

Biochimie 85 (2003) 511-519.

Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.

Evaluation of humoral response to tumor antigens using recombinant expression-based serological mini-arrays (SMARTA).

Lagarkova M.A., Koroleva E.P., Kuprash D.V., Boitchenko V.E., Kashkarova U.A., Nedospasov S.A., Shebzukhov Y.V.

Immunological Letters 85 (2003) 71-74.

Screening of expression cDNA libraries derived from human neoplasms with autologous sera (SEREX) is an established method for defining antigens immunogenic in individual cancer patients. Although the majority of SEREX-derived cDNA clones encode autoantigens, some of them represent shared cancer antigens with cancer-related serological profiles. Routine evaluation of multiple SEREX-derived clones in serological assays using panels of allogeneic sera from cancer patients is an important step towards defining disease parameters of diagnostic and prognostic significance. Here we show how the seroreactivity of multiple SEREX-derived antigens can be simultaneously evaluated using a rapid semi-quantitative protocol of allogeneic screening, which we call SMARTA (serological mini-arrays of recombinant tumor antigens).

Optimisation of dendrimer-mediated gene transfer by anionic oligomers.

Maksimenko A.V., Mandrouguine V., Gottikh M.B., Bertrand J.-R.,

Majoral J.-P., Malvy C.

The Journal of Gene Medicine 5 (2003) 61-71.

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. Their use, however, is limited since they generally lack the efficiency of gene transfer achieved with recombinant viral vectors such as adenovirus. Polyamidoamine (PAMAM) and phosphorus-containing dendrimers (P-dendrimers) are specific polymers with a defined spherical structure. They bind to DNA through electrostatic interactions thus forming complexes that efficiently transfect cells in vitro. The influence of anionic oligomers (oligonucleotides, dextran sulfate) on dendrimer-mediated polyfection of cultured cells has been studied. Anionic oligomers have been found to increase significantly the capacity of the PAMAM and P-dendrimers for DNA delivery into cells when they were mixed with plasmid DNA before addition of dendrimers. The efficiency of the DNA/dendrimer penetration depends on the size, structure and charge of anionic oligomers. Our results represent an important step towards the optimisation of gene transfer mediated by two types of dendrimers. The use of anionic oligomers improves the efficiency of gene expression within cells. As a consequence, a very efficient cell polyfection can be achieved with a lower plasmid quantity for the PAMAM dendrimer greatly increasing the gene expression level for P-dendrimers.

Synthesis and properties of oligodeoxyribonucleotids with mono- and diphosphoryldisulfide internucleotide groupings.

Metelev V.G., Kubareva E.A., Romanova E.A., Oretskaia T.S.

Russian Journal of Bioorganic Chemistry 29 (2003) 57-63.

The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3'-thiophosphorothioates were modified using the thiophosphoryl-disulfide exchange with preactivated 5'-deoxy-5'-mercaptooligonucleotides or 5'-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15-16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.

Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange.

Metelev V.G., Kubareva E.A., Vorob'eva O.V., Romanenkov A.S., Oretskaya T.S.

FEBS Letters 538 (2003) 48-52.

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.

Formation and functioning of fused cholesterol side-chain cleavage enzymes.

Nazarov P.A., Drutsa V.L., Miller W.L., Shkumatov V.M., Luzikov V.N., Novikova L.A.

DNA and Cell Biol. 22 (2003) 243-252.

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.

Gymnophrys cometa and Lecythium sp. are Core Cercozoa: Evolutionary Implications

Nikolaev S.I., Berney C., Fahrni J., Mylnikov A.P., Aleshin V.V., Petrov N.B., Pawlowski J.

Acta Protozool. 42 (2003) 183–190.

Recent phylogenetic analyses based on different molecular markers have revealed the existence of the Cercozoa, a group of .protists including such morphologically diverse taxa as the cercomonad flagellates, the euglyphid testate filose amoebae, the chloroplast-bearing chlorarachniophytes, and the plasmodiophorid plant pathogens. Molecular data also indicate a close relationship between Cercozoa and Foraminifera (Granuloreticulosea). Little is known, however, about the origin of both groups and their phylogenetic relationships. Here we present the complete small-subunit ribosomal RNA (SSU rRNA) sequence of Gymnophrys cometa, formerly included in the athalamid Granuloreticulosea, as well as that of the test-bearing filose amoeba Lecythium sp. Our study shows that the two organisms clearly belong to the Cercozoa, and indicates that Gymnophrys is not closely related to Foraminifera, supporting the view that Granuloreticulosea sensu lato do not form a natural assemblage. Phylogenetic analyses including most available SSU rRNA sequences from Cercozoa suggest that a rigid, external cell envelope appeared several times independently during the evolution of the group. Furthermore, our results bring additional evidence for the wide morphological variety among Cercozoa, which now also include protists bearing granular pseudopodia and exhibiting mitochondria with flattened cristae.

A prior administration of heavy metals reduces thymus lympho-cyte DNA lesions and lipid peroxidation in γ-irradiated mice.

Osipov A.N., Ryabchenko N.I., Ivannik B.P., Dzikovskaya, L.A. Ryabchenko V.I., Kolomijtseva G.Ya.

J. Phys. IV France 107 (2003) 987-992.

In the present work we report that a prior injection of Pb, Cd or Zn salt solutions in SHK male mice decreases the effect followed γ-irradiation on thymus lymphocyte DNA structure and level of lipid peroxidation. It is assumed that the observed phenomenon is caused by activation of protective mechanisms of cells, expression of the genes of antioxidant proteins such as the metallothioneins, etc. Indeed the measurement of malondialdehyde (MDA) in blood plasma showed that the injection of metal salt solutions at median lethal doses a half hour before γ-irradiation (1 Gy) causes the decrease of the MDA contents at 48 h after irradiation on 100% (Zn), 70% (Cd) and 20% (Pb). However we found that combined exposure of the mice also results to significant decrease of the thymus lymphocytes total number of as compared to the irradiation without metals. The elimination of the cells with high level of DNA lesions and existence at least a subset of cells which would survive the current oxidative stress (γ-irradiation) possibly represents one path-way of the survival of individual organism facing stress. In turn the observed decrease of the lesion levels may be reflection of the cell number change.

Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis.

Osipova E.S., Koveza O.V., Troitskij A.V., Dolgikh Yu.I., Shamina Z.B., Gostimskij S.A.

Russian Journal of Genetics 39 (2003) 1412-1419.

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) mark-ers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymor-phism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.

PspGI, a Type II Restriction Endonuclease from the Extreme Thermophile Pyrococcus sp.: Structural and Functional Studies to Investigate an Evolutionary Relationship with Several Mesophilic Restriction Enzymes.

Pingoud V., Conzelmann C., Kinzebach S., Sudina A., Metelev V., Kubareva E., Bujnicki J.M., Lurz R., Lüder G., Xu S.-Y., Pingoud A.

Journal of Molecular Biology 329 (2003) 913-929.

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl2 and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.

Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.

Rubtsova M.P., Sizova D.V., Dmitriev S.E., Ivanov D.S., Prassolov V.S., Shatsky I.N.

J. Biol. Chem. 278 (2003) 22350-2256.

A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.

Analysis of the genetic structure of northwestern bering sea walleye pollock, Theragra chalcogramma.

Shubina E.A., Melnikova M.N., Glubokov A.I., Mednikov B.M.

Enviromental Biology of Fishes 68 (2003) 451-459.

The intraspecific structute of major populations of the walleye pollock Theragra сhalcogramma from the North-Western part of the Bering sea is studied. Specimens from Navarin, Olyutorsky and Shirshov shoals were sampled in spawning season 2001. Preliminary claster analysis (TREECON) of PCR-RAPD data revealed the existence of cluster with low level of bootstrap supporting, which generally correspond to geographic localization of the shoals. Processing of microsatellite sequence data (loci Tch12, Tch14, Tch15 and Tch18) with GENEPOP demonstrated the deviation from HWE and differentiation between the samples. The value of the interpopulation variance (F_st = 0.02) corresponded to published data on marine stocks, which were subject to high levels of gene flow. The Shirshov group was found to be equidistant from the Navarin and Olyutor groups, with the genetic distance between the latter two being significantly less. Analysis of the microsatellite loci inferred disparities in their selective capacities. Loci Tch12 and Tch15 are found to be highly homozigous with low levels of polymorphism. Tch12 clearly follows the “3 band” pattern. Tch15 shows sign of linkage disequilibrium. Comparison of Fst values for the loci with the mean standardized variance f₀ suggests that Tch12 is under pressure of disruptive selection, while Tch15 is rather neutral and Tch14 and Tch18 are under the influence of balance selection. As well the disscussion is conducted in the context of undeterminate group migration.

Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups.

Taranenko M., Rykhlevskaya A., Mtchedlidze M., Laval J., Kuznetsova S.

European Journal of Biochemistry 270 (2003) 2945-2049.

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.

Crosslinking of Cys142 of Methyltransferase SsoII with DNA Duplexes Containing a Single Internucleotide Phosphoryldisulfide Link.

Vorob’eva O.V., Romanenkov A.S., Metelev V.G., Karyagina A.S., Lavrova N.V., Oretskaya T.S., Kubareva E.A.

Molecular Biology (Moscow) 37 (2003) 772-779.

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (.SsoII). The possibility of duplex–.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of .SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA–enzyme complex.

Apoptosis in the initial leaf of etiolated wheat seedlings: influence of the antioxidant ionol (BHT) and peroxides.

Zamyatnina V.A., Bakeeva L.E., Aleksandrushkina N.I., Vanyushin B.F.

Biochemistry (Moscow) 67 (2002) 212-221.

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva L. E. et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27 x 10(-4) M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H2O2, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.

Synthesis of 2'-modified oligonucleotides containing aldehyde or ethylenediamine groups.

Zatsepin T.S., Romanova E.A., Stetsenko D.A., Gait M.J., Oretskaya T.S.

Nucleosides Nucleotides Nucleic Acids. 22 (2003) 1383-1385.

Oligonucleotides carrying 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2'-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2'-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.

PSF Acts through the Human Immunodeficiency Virus Type 1 mRNA Instability Elements To Regulate Virus Expression.

Zolotukhin A.S., Michalowski D., Bear J., Smulevitch S.V., Traish A.M., Peng R., Patton J., Shatsky I.N., Felber B.K.

Molecular and Cellular Biology 23 (2003) 6618-6630.

Human immunodeficiency virus type 1 (HIV) gag/pol and env mRNAs contain cis-acting regulatory elements (INS) that impair stability, nucleocytoplasmic transport, and translation by unknown mechanisms. This downregulation can be counteracted by the viral Rev protein, resulting in efficient export and expression of these mRNAs. Here, we show that the INS region in HIV-1 gag mRNA is a high-affinity ligand of p54nrb/PSF, a heterodimeric transcription/splicing factor. Both subunits bound INS RNA in vitro with similar affinity and specificity. Using an INS-containing subgenomic gag mRNA, we show that it specifically associated with p54nrb in vivo and that PSF inhibited its expression, acting via INS. Studying the authentic HIV-1 mRNAs produced from an infectious molecular clone, we found that PSF affected specifically the INS-containing, Rev-dependent transcripts encoding Gag-Pol and Env. Both subunits contained nuclear export and nuclear retention signals, whereas p54nrb was continuously exported from the nucleus and associated with INS-containing mRNA in the cytoplasm, suggesting its additional role at late steps of mRNA metabolism. Thus, p54nrb and PSF have properties of key factors mediating INS function and likely define a novel mRNA regulatory pathway that is hijacked by HIV-1.

1,2-Diol and hydrazide phosphoramidites for solid-phase synthesis and chemoselective ligation of 2'-modified oligonucleotides.

Zubin E.M., Stetsenko D.A., Oretskaya T.S., Gait M.J.

Nucleosides Nucleotides Nucleic Acids. 22 (2003) 1375-1378.

The preparation of two novel 2'-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2'-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO4 to generate 2'-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions.

VI. Enzymology and biotechnology

The activation of glycolysis performed by the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the model system

Arutyunov D.Y., Muronetz V.I.

Biochemical and Biophysical Research Communications 300 (2003) 149-154.

Influence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) on glycolysis was investigated. The addition of GAPN-which oxidizes glyceraldehyde-3-phosphate directly to the 3-phosphoglyceric acid-led to the strong increase in the rate of lactate accumulation in the rat muscle extract with low ADP content. The lactate accumulation was also observed in the presence of GAPN in rat muscle extract, which contained only ATP and no ADP. This can be the evidence of the "futile cycle" stimulated by GAPN. Here ADP can be regenerated from ATP by the phosphoglycerate kinase reaction. The high resistance of GAPN from Streptococcus mutans towards inactivation by natural oxidant-H₂O₂ was showed. This feature distinguishes GAPN from phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is very sensitive to modification by hydrogen peroxide. A possible role of the oxidants and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the regulation of glycolysis is discussed.

Oxidation of glyceraldehyde-3-phosphate dehydrogenase enhances its binding to nucleic acids

Arutyunova E.I., Danshina P.V., Domnina L.V., Pleten A.P., Muronetz V.I.

Biochemical and Biophysical Research Communications 307 (2003) 547-552.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with various activities far from its enzymatic function. Here, we showed that the oxidation of SH-groups of the active site of GAPDH enhanced its binding with total transfer RNA or with total DNA. Both NAD and NADH-the cofactors of GAPDH-inhibited the GAPDH-RNA (DNA) interaction, though NAD was much less effective than NADH in the case of oxidized GAPDH. Oxidation of GAPDH strongly decreased its affinity to NAD but not to NADH. Immobilized tetramers of GAPDH dissociated into dimers during the incubation with total RNA but not DNA. The staining of HeLa cells with monoclonal antibodies specific to dimers, monomers or the denatured form of GAPDH revealed the condensation of non-native forms of GAPDH in the nucleus. The role of oxidation of GAPDH in the regulation of the quaternary structure of the enzyme and in its interaction with nucleic acids is discussed.

Extracts of lung cancer cells reveal antitumour antibodies in sera of patients with lung cancer

Bazhin A.V., Savchenko M.S., Shifrina O.N., Chikina S.Y., Goncharskaia M.A., Jaques G, Chuchalin A.G., Philippov P.P.

Eur. Respir. J. 21 (2003) 342-346.

The objective of the present study was to reveal antitumour antibodies in sera of patients with small cell lung cancer (SCLC). The antibodies in sera of patients with SCLC and other tumours were detected by immunoblotting with whole extracts of SCLC cells as the antigen source. Sera of patients with various pulmonological disorders, irradiated during the liquidation of consequences of the Chernobyl nuclear power plant incident (a high-risk group in lung cancer), were also analysed. The present authors' found that SCLC sera contain a set (pattern) of antitumour antibodies which are rarely detected in sera of patients with cancers different from SCLC and very rarely, if ever, present in sera of healthy individuals. The sensitivity and the specificity of the pattern are equal to 80% and 91%, correspondingly. In the high-risk group in lung cancer, the frequencies of the antibodies are somewhat lower than the corresponding values in SCLC sera, but significantly larger than those in healthy sera. The findings of the present study create a basis for clinical application of the antitumour antibodies described.

2-Oxo acid dehydrogenase complexes in redox regulation.

Bunik V.I.

European Journal of Biochemistry 270 (2003) 1036-1042.

A number of cellular systems cooperate in redox regulation, providing metabolic responses according to changes in the oxidation (or reduction) of the redox active components of a cell. Key systems of central metabolism, such as the 2-oxo acid dehydrogenase complexes, are important participants in redox regulation, because their function is controlled by the NADH/NAD⁺ ratio and the complex-bound dihydrolipoate/lipoate ratio. Redox state of the complex-bound lipoate is an indicator of the availability of the reaction substrates (2-oxo acid, CoA and NAD⁺) and thiol-disulfide status of the medium. Accumulation of the dihydrolipoate intermediate causes inactivation of the first enzyme of the complexes. With the mammalian pyruvate dehydrogenase, the phosphorylation system is involved in the lipoate-dependent regulation, whereas mammalian 2-oxoglutarate dehydrogenase exhibits a higher sensitivity to direct regulation by the complex-bound dihydrolipoate/lipoate and external SH/S-S, including mitochondrial thioredoxin. Thioredoxin efficiently protects the complexes from self-inactivation during catalysis at low NAD⁺. As a result, 2-oxoglutarate dehydrogenase complex may provide succinyl-CoA for phosphorylation of GDP and ADP under conditions of restricted NAD⁺ availability. This may be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool. Concomitantly, thioredoxin stimulates the complex-bound dihydrolipoate-dependent production of reactive oxygen species. It is suggested that this side-effect of the 2-oxo acid oxidation at low NAD⁺ in vivo would be overcome by cooperation of mitochondrial thioredoxin and the thioredoxin-dependent peroxidase, SP-22.

Resolution of (RS)-phenylglycinonitrile by penicillin acylase-catalyzed acylation in aqueous medium

Chilov G.G., Moody H.M., Boesten W.H.J, Švedas V.K.

Tetrahedron: Asymmetry 14 (2003) 2613-2617.

A new strategy for the biocatalytic resolution of (R,S)-phenylglycinonitrile, a crucial intermediate in the antibiotic industry, has been developed. While former techniques exploit nitrilases or combinations of nitrile hydratases and amidases, manipulating with nitrile functionality, the current approach is based on a highly efficient and enantioselective acylation of the -amino group with phenylacetic acid catalyzed by a well known enzyme, penicillin acylase from E. coli, in slightly acidic aqueous medium. It is shown that since the condensation product is poorly soluble, removal of (S)-phenylglycinonitrile from the reaction sphere is almost complete and irreversible, favoring kinetics of the process and making high conversion possible. The proposed approach is characterized by high space-time yield and extends the scope of enzymatic synthesis in aqueous medium.

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Dan'shina P.V., Schmalhausen E.V., Arutiunov D.Y., Pleten' A.P., Muronetz V.I.

Biochemistry (Moscow) 68 (2003) :593-600.

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.

Influence of Complexing Polyanions on the Thermostability of Basic Proteins

Ivinova O.N., Izumrudov V.A., Muronetz V.I., Galaev I.Yu., Mattiasson B.

Macromol. Biosci. 3 (2003) 210–215.

The results obtained in the present study indicate the ability of the polyanions to significantly reduce the onset of thermodenaturation of proteins without causing noticeable changes in their enzymatic activity over a wide temperature range below the critical temperature at which denaturation starts. This finding appears to be crucial for the further deve-lopment of immobilized enzymes and is essential for under-standing how proteins function when they are immobilized inside charged matrices in vivo.

Penicillin acylase-catalyzed peptide synthesis in aqueous medium: a chemo-enzymatic route to stereoisomerically pure diketopiperazines

Khimiuk A.Y., Korennykh A.V., van Langen L.M., van Rantwijk F., Sheldon R.A., Švedas V.K.

Tetrahedron: Asymmetry 14 (2003) 3123-3128.

A range of non-natural dipeptides of the general formula -(-)-phenylglycyl--X, where X is a natural -amino acid, have been prepared by penicillin acylase-catalyzed synthesis in aqueous medium from -(-)-phenylglycine amide and the corresponding amino acids. The conversion of the dipeptides to the corresponding dipeptide esters, followed by their subsequent spontaneous cyclization afforded the corresponding stereoisomerically pure diketopiperazines.

Induced absorption band of holotransketolase and its interpretation.

Kovina M.V., Bykova I.A., Meshalkina L.E., Kochetov G.A.

Biochemistry (Moscow) 68 (2003) 247-251.

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band. Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps. As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown. The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids. They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme. However none of these hypotheses had been substantiated experimentally. According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1;-atom of the ThDP aminopyrimidine ring to Glu₄₁₈, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe₄₄₅.

New Photoreactive Cleavable Reagents with Trifluoromethyldiazirine Group.

Mchedlidze M.T., Sumbatyan N.V., Bondar D.A., Taranenko M.V., Korshunova G.A.

Russian Journal of Bioorganic Chemistry 29 (2003) 194-201.

Photoreactive crosslinking reagents that simultaneously contain a trifluoromethyldiazirine and an o-nitrobenzyl groups were synthesized for the first time. Photochemical properties of the reagents were studied, and the possibility of separate activation of the diazirine group and o-nitrobenzyl linker was shown.

Isolation of antigens and antibodies by affinity chromatography.

Muronetz V.I., Korpela T.

J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 790 (2003) 53-66.

Antibody-antigen binding constants are commonly strong enough for an effective affinity purification of antibodies (by immobilized antigens) or antigens (by immobilized antibodies) to work out a straightforward purification method. A drawback is that antibodies are large protein molecules and subject to denaturation under conditions required for the elution from the complex. Structures of antigens can vary but usually antigens are also equally subject to similar problems. The lability of the components can sometimes make the procedure sophisticated, but usually in all cases it is possible to find a satisfactory approach. In certain cases, specific interactions of the Fc part of antibodies are more facile to exploit for their purification.

Interdomain communications in bifunctional enzymes: how are different activities coordinated?

Nagradova N.

IUBMB Life 55 (2003) 459-466

Although bifunctional enzymes containing two different active centers located within separate domains are quite common in living systems, the significance of this bifunctionality is not always clear, and the molecular mechanisms of site-site interactions in such complex systems have come under the scrutiny of science only in recent years. This review summarizes recent data on the mechanisms of communication between active centers in bifunctional enzymes. Three types of enzymes are considered: (1) those catalyzing consecutive reactions of a metabolic pathway and exhibiting substrate channeling (glutamate synthase and imidazole glycerol phosphate synthase), (2) those catalyzing consecutive reactions without substrate channeling (lysine-ketoglutarate reductase/saccharopine dehydrogenase), and (3) those catalyzing opposed reactions (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase). The functional role of interdomain communications is briefly discussed.

The COOH termini of NBC3 and the 56-kDa H⁺-ATPase subunit are PDZ motifs involved in their interaction.

Pushkin A., Abuladze N., Newman D., Muronets V., Sassani P., Tatishchev S., Kurtz I.

Am. J. Physiol. Cell Physiol. 284 (2003) C667-C673.

The electroneutral sodium bicarbonate cotransporter 3 (NBC3) coimmunoprecipitates from renal lysates with the vacuolar H⁺-ATPase. In renal type A and B intercalated cells, NBC3 colocalizes with the vacuolar H⁺-ATPase. The involvement of the COOH termini of NBC3 and the 56-kDa subunit of the proton pump in the interaction of these proteins was investigated. The intact and modified COOH termini of NBC3 and the 56-kDa subunit of the proton pump were synthesized, coupled to Sepharose beads, and used to pull down kidney membrane proteins. Both the 56- and the 70-kDa subunits of the proton pump, as well as a PDZ domain containing protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1), were bound to the intact 18 amino acid NBC3 COOH terminus. A peptide truncated by five COOH-terminal amino acids did not bind these proteins. Replacement of the COOH-terminal leucine with glycine blocked binding of both the proton pump subunits but did not affect binding of NHERF-1. The 18 amino acid COOH terminus of the 56-kDa subunit of the proton pump bound NHERF-1 and NBC3, but the truncated and modified peptide did not. A complex of NBC3, the 56-kDa subunit of the proton pump, and NHERF-1 was identified in rat kidney. The data indicate that the COOH termini of NBC3 and the 56-kDa subunit of the vacuolar proton pump are PDZ-interacting motifs that are necessary for the interaction of these proteins. NHERF-1 is involved in the interaction of NBC3 and the vacuolar proton pump.

Antirecoverin autoantibodies in the patient with non-small cell lung cancer but without cancer-associated retinopathy.

Savchenko M.S., Bazhin A.V., Shifrina O.N., Demoura S.A., Kogan E.A., Chuchalin A.G., Philippov P.P.

Lung Cancer 41 (2003) 363-367.

The goal of the present study was to analyze serum and tumor tissue of a patient with non-small cell lung cancer (NSCLC) for the presence of autoantibodies against recoverin (anti-Rc) and recoverin expression, correspondingly. Using immunoblotting with recombinant recoverin as an antigen, we have detected anti-Rc in serum of the patient. At the same time, the patient did not manifest any signs of cancer-associated retinopathy (CAR). Polyclonal (monospecific) antibodies against recoverin used for immunohistochemical analysis of the patient's tumor revealed recoverin expression in the tumor sections. To our knowledge, this is the first case of the presence of serum anti-Rc in NSCLC patients in the absence of paraneoplastic retina degeneration.

Functional restoration of the Ca²⁺-myristoyl switch in a recoverin mutant.

Senin I.I., Vaganova S.A., Weiergraber O.H., Ergorov N.S., Philippov P.P., Koch K.W.

Journal of Molecular Biology 330 (2003) 409-418.

Recoverin is a neuronal calcium sensor protein that plays a crucial role in vertebrate phototransduction. It undergoes a Ca²⁺-myristoyl switch when Ca²⁺ binds to its two functional EF-hand motifs (EF-hands 2 and 3), each present in one of recoverin's two domains. Impairment of Ca²⁺-binding in recoverin leads to a disturbance of the Ca²⁺-myristoyl switch and loss of its regulatory properties, i.e. inhibiton of rhodopsin kinase. We have engineered recoverin mutants with either of the two functional EF-hands disabled, but with a functional Ca²⁺-binding site in EF-hand 4. While a defect in EF-hand 2 could not be rescued by the additional EF-hand 4, the impairment of EF-hand 3 was powerfully compensated by Ca²⁺-binding to EF-hand 4. For example, the myristoylated form of the latter mutant bound to membranes in a Ca²⁺-dependent way and was able to inhibit rhodopsin kinase in a way similar to that of the wild-type protein. Thus, for recoverin to undergo a Ca²⁺-myristoyl switch, it is necessary and sufficient to have either of the two EF-hands in the second domain in a functional state. On the basis of these results and inspection of published three-dimensional structures of recoverin, we propose a model highlighting the mutual interdependence of sterical configurations in EF-hands 3 and 4 of recoverin.

Arachidonic acid in astrocytes blocks Ca²⁺ oscillations by inhibiting store-operated Ca²⁺ entry, and causes delayed Ca²⁺ influx.

Sergeeva M., Strokin M., Wang H., Ubl J.J., Reiser G.

Cell Calcium 33 (2003) 283-292.

ATP-elicited oscillations of the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca²⁺ store refilling. Short-term application of AA, but not ETYA, blocked Ca²⁺ influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca²⁺]_i oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca²⁺]_i increase. This AA-induced [Ca²⁺]_i rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 M Gd³⁺, indicative for the influx of extracellular Ca²⁺. Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca²⁺]_i, firstly, a rapid reduction of capacitative Ca²⁺ entry thereby suppressing [Ca²⁺]_i oscillations, and secondly inducing a delayed activation of Ca²⁺ entry, also sensitive to low Gd³⁺ concentration.

Ascorbate-induced oxidation of glyceraldehyde-3-phosphate dehydrogenase.

Schmalhausen E.V., Pleten' A.P., Muronetz V.I.

Biochemical and Biophysical Research Communications 308 (2003) 492-496.

Oxidation of the essential cysteins of glyceraldehyde-3-phosphate dehydrogenase into the sulfenic acid derivatives was observed in the presence of ascorbate, resulting in a decrease in the dehydrogenase activity and the appearance of the acylphosphatase activity. The oxidation was promoted by EDTA, NAD⁺, and phosphate, and blocked in the presence of deferoxamine. The ascorbate-induced oxidation was suppressed in the presence of catalase, suggesting the accumulation of hydrogen peroxide in the conditions employed. The data indicate the metal-mediated mechanism of the oxidation due to the presence of metal traces in the reaction medium. Physiological importance of the mildly oxidized GAPDH is discussed in terms of its ability to uncouple glycolysis and to decrease the ATP level in the cell.

Studies OF THIAMIN DIPHOSPHATE BINDING TO THE YEAST APOTRANSKETOLASE.

Selivanov V.A., Kovina M.V., Kochevova N., Meshalkina L.E., Kochetov G.A.

J. Mol. Catalysis B. Enzymatic. 26 (2003) 33-40.

Previously it was shown that the binding of thiamin diphosphate proceeds through two steps: fast primary binding and the subsequent slow conformational transition of the apoprotein. In the presence of Ca²⁺, the coenzyme binding occurs with negative cooperativity – owing to the increased rate of the reverse conformational transfer in one of the active centers after completion of ThDP binding at both active centers. There are three viewpoints on the enzyme behavior upon substitution of Mg²⁺ for Ca²⁺: (a) negative cooperativity between the two centers is retained; (b) turns positive; (c) totally disappears. In this work, a comparative investigation of the interaction between ThDP and apotransketolase was undertaken and the negative cooperativity between the two centers in the presence of Mg²⁺ , just as in the presence of Ca²⁺, was demonstrated - albeit with the former cation it was somewhat less pronounced. The negative cooperativity with Mg²⁺, just with Ca²⁺, was caused by an increase in the rate of reverse conformational transfer after the ThDP binding completion in both active centers.

Effectory Site in Escherichia coli Inorganic Pyrophosphatase is Revealed Upon Mutation at the Intertrimeric Interface.

Sitnik T.S., Vainonen J.P., Rodina E.V., Nazarova T.I., Kurilova S.A., Vorobyeva N.N., Avaeva S.M.

IUBMB Life 55 (2003) 37-41.

Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPP_i hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PP_i binding at an effectory subsite. Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPP_i hydrolysis though its magnesium complex is a competitive inhibitor. The Asp26Ala variant is the first example of hexameric E-PPase demonstrated to have an activatory subsite.

Force field parametrization for 6-aminopenicillanic acid.

Stroganov O.V., Chilov G.G., Švedas V.K.

Journal of Molecular Structure: THEOCHEM 631 (2003) 117-125.

A complete set of AMBER force field parameters for 6-aminopenicillanic acid, crucial moiety of -lactam antibiotics, has been developed. Equilibrium geometry was derived from RHF/6-31G* calculations, stretching constants were computed from quantum mechanical hessian, partial atomic charges were assigned according to restrained electrostatic potential fit methodology. Torsional parameters were also derived from quantum mechanical calculations. It was shown that unusual chemical structure of 6-aminopenicillanic acid with two merged four- and five-member rings has found its reflection in the set of molecular mechanical parameters: a number of bonds and angles appeared to be much more rigid than those described by classic AMBER field. The quality of derived set parameters was attested by computing molecular geometry, spectroscopic and thermodynamic properties.

Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca²⁺

Strokin M., Sergeeva M., Reiser G.

British Journal of Pharmacology 139 (2003)1014-1022.

Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. Primary astrocytes were cultured under standard conditions and prelabeled with [¹⁴C]DHA or with [³H]AA. Adenosine 5'-triphosphate (ATP) (20 µM applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂), since it is blocked by the selective iPLA₂ inhibitor 4-bromoenol lactone (BEL, 5 µM) and is not affected either by removal of Ca²⁺ from extracellular medium or by suppression of intracellular Ca²⁺ release through PLC inhibitor (U73122, 5 µM). AA release, on the other hand, which is stimulated by ATP, is attributed to Ca²⁺-dependent cytosolic PLA₂ (cPLA₂). AA release is abolished by U73122 and, by removal of extracellular Ca²⁺, is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 µM), a general inhibitor of intracellular PLA₂ s. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA₂ and 40 kDa protein reactive to iPLA₂ antibodies. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 µM), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 µM) and inhibited by 34% by H89 (10 µM), inhibitors of adenylyl cyclase and PKA, respectively. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. Different sensitivities of iPLA₂ and cPLA₂ to Ca²⁺ and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

Activation of NF-kappa B transcription factor in human neutrophils by sulphatides and L-selectin cross-linking.

Turutin D.V., Kubareva E.A., Pushkareva M.A., Ullrich V., Sud'ina G.F.

FEBS Letters 536 (2003) 241-245.

Sulphated galactocerebroside (sulphatide) has been established as a ligand for L-selectin and shown to trigger intracellular signals in human neutrophils. We have found that sulphatide activated transcription factor NF-kappa B in human neutrophils in a concentration-dependent manner whereas non-sulphated galactocerebroside did not demonstrate such an effect. The activation was inhibitable by pretreatment with primary monoclonal anti-L-selectin antibody (clone LAM1-116). Binding of the primary antibody to L-selectin was insufficient to induce NF-kappa B activation but cross-linking of L-selectin with a secondary antibody was effective. α -Chymotrypsin, the agent known to shed L-selectin, activated NF-kappa B by itself. The response to sulphatides was inhibited by jasplakinolide, an actin-polymerising agent known to downregulate surface expression of L-selectin, Fc gamma RIIIb, CD43 and CD44. Recently we have reported that sulphatide stimulated the attachment of human neutrophils to collagen via Mac1 (CD11b/CD18) integrin [Sud'ina et al., Biochem. J. 359 (2001) 621-629]. We now show signalling from sulphatide to NF-kappa B activation and discuss its involvement in neutrophil adhesion.

Active Dimeric Form of Inorganic Pyrophosphatase from Escherichia coli.

Vainonen Y.P., Vorobyeva N.N., Kurilova S.A., Nazarova T.I., Rodina E.V., Avaeva S.M.

Biochemistry (Moscow) 68 (2003) 1195-1199.

A dimeric form can be obtained from native hexameric Escherichia coli inorganic pyrophosphatase (E-PPase) by destroying the hydrophobic intersubunit contacts, and it has been shown earlier to consist of the subunits of different trimers. The present paper is devoted to the kinetic characterization of such a "double-decked" dimer obtained by the dissociation of either the native enzyme or the mutant variant Glu145Gln. The dimeric form of the native inorganic pyrophosphatase was shown to retain high catalytic efficiency that is in sharp contrast to the dimers obtained as a result of the mutations at the intertrimeric interface. The dimeric enzymes described in the present paper, however, have lost the regulatory properties, in contrast to the hexameric and trimeric forms of the enzyme.

Peptide derivatives of antibiotics tylosin and desmycosin, protein synthesis inhibitors.

Sumbatyan NV, Korshunova GA, Bogdanov AA.

Biochemistry (Moscow) 68 (2003) 1156-1158.

Biologically active peptide derivatives of 16-member macrolide antibiotics were synthesized as potential probes for the investigation of nascent peptide chain topography in the ribosomal exit tunnel. The tylosin and desmycosin aldehyde groups at the C6 position of the lactone ring were modified by the aminooxyacetyl-L-alanyl-L-alanine methyl ester.

A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction.

Vrzheshch P.V., Batanova E.A., Mevkh A.T., Varfolomeev S.D., Gazaryan I.G., Thorneley R.N.

Biochem. J. 372 (2003) 713-724.

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector 'kinetic connectivities' between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H₂O₂. To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I-adrenaline complex.

Study of nucleophile binding in the penicillin acylase active center. Kinetic analysis.

Youshko M.I., Bukhanov A.L., Svedas V.K.

Biochemistry (Moscow) 68 (2003) 334-338.

The influence of the external nucleophile (6-aminopenicillanic acid) on the kinetics of the penicillin acylase-catalyzed acyl transfer reactions was studied using a highly sensitive spectrophotometric assay. An adequate kinetic scheme is suggested based on kinetic analysis of the experimental dependencies of the k_cat and K_m values on the nucleophile concentration. The proposed kinetic scheme has been verified by a quantitative description of the above-mentioned experimental dependencies using the set of kinetic parameters obtained from independent experiments. Such an approach can be used for modeling of different penicillin acylase-catalyzed acyl transfer reactions.

Detection of annexin IV in bovine retinal rods.

Zernii E.Y., Tikhomirova N.K., Philippov P.P., Senin I.I.

Biochemistry (Moscow) 68 (2003) 129-160.

The fraction of proteins capable of binding to photoreceptor membranes in a Ca²⁺-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.

Rho-dependent formation of epithelial "leader" cells during wound healing.

Omelchenko T., Vasiliev J.M., Gelfand I.M., Feder H.H., Bonder E.M.

Proc. Natl. Acad. Sci. USA 100 (2003) 10788–10793.

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special "leader" cells at the tops of outgrowths and "follower" cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.