Документ взят из кэша поисковой машины. Адрес оригинального документа : http://www.genebee.msu.ru/ann2002.html
Дата изменения: Fri Feb 28 14:51:09 2003
Дата индексирования: Mon Oct 1 19:34:47 2012
Кодировка: Windows-1251
I


M.V. Lomonosov Moscow State University 



A.N. Belozersky Institute

of Physico-Chemical Biology



ANNUAL REPORT

2002


CONTENT

  Biology of cell and cell organelles

  Bioenergetics and photosynthesis

  Mathematical models in biology

  Molecular virology

  Structure, expression and evolution of genom

  Enzymology and biotechnology


I. BIOLOGY OF CELL AND CELL ORGANELLES

Modifications of vestibular responses of individual reticulospinal neurons in lamprey caused by unilateral labyrinthectomy

Deliagina T.G., Pavlova E.L.

Journal of Neurophysiology 87 (2002) 1-14

A postural control system in the lamprey is driven by vestibular input and maintains the dorsal-side-up orientation of the animal during swimming. After a unilateral labyrinthectomy (UL), the lamprey continuously rolls toward the damaged side. Normally, a recovery of postural equilibrium ("vestibular compensation") takes about 1 mo. However, illumination of the eye contralateral to UL results in an immediate and reversible restoration of equilibrium. Here we used eye illumination as a tool to examine a functional recovery of the postural network. Important elements of this network are the reticulospinal (RS) neurons, which are driven by vestibular input and transmit commands for postural corrections to the spinal cord. In this study, we characterized modifications of the vestibular responses in individual RS neurons caused by UL and the effect exerted on these responses by eye illumination. The activity of RS neurons was recorded from their axons in the spinal cord by chronically implanted electrodes, and spikes in individual axons were extracted from the population activity signals. The same neurons were recorded both before and after UL. Vestibular stimulation (rotation in the roll plane through 360 degrees ) and eye illumination were performed in quiescent animals. It was found that the vestibular responses on the UL-side changed only slightly, whereas the responses on the opposite side disappeared almost completely. This asymmetry in the bilateral activity of RS neurons is the most likely cause for the loss of equilibrium in UL animals. Illumination of the eye contralateral to UL resulted, first, in a restoration of vestibular responses in the neurons inactivated by UL and in an appearance of vestibular responses in some other neurons that did not respond to vestibular input before UL. These responses had directional sensitivity and zones of spatial sensitivity similar to those observed before UL. However, their magnitude was smaller than before UL. Second, the eye illumination caused a reduction of the magnitude of vestibular responses on the UL side. These two factors tend to restore symmetry in bilateral activity of RS neurons, which is the most likely cause for the recovery of equilibrium in the swimming UL lamprey. Results of this study are discussed in relation to the model of the roll control system proposed in our previous studies as well as in relation to the vestibular compensation.

A major nucleolar protein B23 as a marker of proliferation activity of human peripheral lymphocytes 

Dergunova N.N., Bulycheva T.I., Artemenko E.G., Shpakova A.P., Pegova A.N., Gemjian E.G., Dudnik O.A., Zatsepina O.V., Malashenko O.S. 

Immunology Letters 83 (2002) 67-72 

A novel monoclonal antibody (Mab) (called 3C9) against a major nucleolar phosphoprotein B23 was used to study 1323 qualitative and quantitative alterations in phytohemagglutinin (PHA) - stimulated human peripheral blood lymphocytes in indirect immunofluorescence and Western blots. It was shown that lymphocyte proliferation was accompanied by gradual augmentation of nucleoli and their accumulation of the protein B23 tip to 2-fold by 16 h and 40-50 fold by 72 h. as compared with the non-stimulated cells. By parallel immunolabeling with the anti-Ki-67 antibody. it was shown that the early changes of 1323 amount and localization occurred before an appearance of Ki-67 protein, a ell-known marker of proliferating cells. Our results evidence that antibodies against B23 might be applied for recognition of human peripheral lymphocytes at early stages of their activation for proliferation, preceding the S-phase. 

Effects of transmembrane gradients of bicarbonate and ammonium on junctional and nonjunctional membrane conductances in BHK cells 

Dunina-Barkovskaya A.Y., Bujurina I.M., Pivovarov V.S., Frolov V.A.

Membrane and Cell Biology 14 (2001) 791-811

Weak acids are efficient blockers of gap-junctional conductance. It is generally accepted that intracellular acidification produced by weak acids fully accounts for the gap-junctional uncoupling. Protonation of the cytoplasmic portions of the channel-forming protein connexin is thought to lead to the conformational changes switching the channel from the open into the closed state. If this is the only mechanism of the weak-acid induced uncoupling, then the correlation between junctional conductance (Gj) and intracellular pH (pHin) should not depend on the means of intracellular acidification. We compared the responses of junctional conductance in BHK cells measured in double whole-cell experiments to the applied transmembrane concentration gradients of bicarbonate or ammonium. These treatments were to lower pHin in a predictable way according to the equations: pHin = pHout -lg[[HCO3]out/HCO3-]in) or pHin = PHout - lg[[NH4+]in[NH4+]out), respectively. We found that the behavior of Gj depended on the substance used. At a 500-fold bicarbonate gradient (calculated pHin approximately 4.8) the cells remained coupled, while a 100- or 10-fold gradient of ammonium imposing pHin approximately 6.1 produced fast uncoupling. The responses of junctional conductance were often accompanied or preceded by changes of non-junctional membrane conductance. We suggest that the mechanisms of the weak acid/base-induced channel gating may contain an additional "lipophilic" component due to the presence of the non-dissociated form of the acid/base in cell membrane.

Cytoskeletal Mechanisms of the Formation of Heterocellular Contacts 

Fetisova E.K., Ivanova O.Yu., Omelchenko T., Vasiliev Ju.M.

Russian Journal of Developmental Biology 33 (2002) 306-310

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella. No changes were observed in the actin organization of the fibroblast. In architecture, the heterophilic adhesion contacts resembled the contacts between fibroblasts. Both heterophilic and homophilic contacts were transient, rather than constant structures. The formation of heterophilic contacts in mixed cultures can serve as a model of formation of a tissue system consisting of epithelium and mesenchyme.

Mode of epithelium-fibroblast interaction in mixed heterotypic cell cultures

Fetisova E.K., Ivanova O.Iu., Vasil'ev Iu.M.

Tsitologiia 44 (2002) 235-241

The interaction between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts (diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic cell cultures. The mode of cell interaction depends on the manner of their collision. At collision of the epithelium lamella and the lateral side of fibroblast, the lamella was seen to creep under the lateral side to force back the fibroblast. At the frontal collision of epithelium and fibroblast lamellae, the mode of interaction depends on the local situation. With the presence of a free substratum around, the fibroblast formed a new lamella and moved aside from the place of collision. In the case, when the neighboring cells prevented fibroblast from moving, it migrated under the epithelium. In this work, we have first demonstrated the formation of specialized intercellular adhesions between epithelium and fibroblasts. The cultures were studied by phase contrast, interference reflection or video tape recording, using an image processing system (Hamamatsu). For studying adhesion, immuno-fluorescent methods were performed.

Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and is inducible NO synthase dependent 

Kapinya K.J., Lowl D., Futterer C., Maurer M., Waschke K.F., Isaev N.K., Dirnagl U. 

Stroke 33 (2002) 1889-1898

Background and Purpose-We tested whether volatile anesthetics induce neuroprotection that is maintained for a prolonged time. Methods-Rats were pretreated for 3 hours with 1 minimal anesthetic concentration of isoflurane or halothane in normal air (anesthetic preconditioning [AP]). The animals were subjected to permanent middle cerebral artery occlusion (MCAO) at 0, 12, 24, or 48 hours after AP. Halothane-pretreated animals were subjected to MCAO 24 hours after AP. Histological evaluation of infarct volumes was performed 4 days after MCAO. Cerebral glucose utilization was measured 24 hours after AP with isoflurane. Primary cortical neuronal cultures were exposed to 1.4% isoflurane for 3 hours. Oxygen-glucose deprivation (OGD) was performed 24 hours after AP. Injury was assessed 24 hours later by measuring the release of lactate dehydrogenase into the medium 24 hours after OGD. Results- Isoflurane anesthesia at 0, 12, and 24 hours before MCAO or halothane anesthesia 24 hours before MCAO significantly reduced infarct volumes (125 + 42 mm3, P=0.024; 118 + 51 mm3, P=0.008; 120 + 49 mm3, P=0.009; and 121 + 48 mm3, P=0.018, respectively) compared with control volumes (180 + 51 mm3). Three hours of isoflurane anesthesia in rats did not have any effect on local or mean cerebral glucose utilization measured 24 hours later. Western blot analysis from cortical extracts of AP-treated animals revealed an increase of the inducible NO synthase (iNOS) protein beginning 6 hours after AP. The iNOS inhibitor aminoguanidine (200 mg/kg IP) eliminated the infarct-sparing effect of AP. In cultured cortical neurons, isoflurane exposure 24 hours before OGD decreased the OGD- induced release of lactate dehydrogenase by 49% (P=0.002). Conclusions-Pretreatment with volatile anesthetics induces prolonged neuroprotection in vitroand in vivo, a process in which iNOS seems to be critically involved. 

Life cycle of MTs: persistent growth in the cell interior, asymmetric transition frequencies and effects of the cell boundary 

Komarova Y.A., Vorobjev I.A., Borisy G.G. 

Journal of Cell Science 115 (2002) 3527-3539

Microtubule dynamics were investigated in CHO and NRK cells by novel experimental approaches designed to evaluate the microtubule behavior in the cell interior. These, approaches were: (1) laser photobleaching of a path through the centrosome; (2) direct observation of microtubules in centrosome-containing cytoplasts; (3) GFP-CLIP-170 expression as a marker for microtubule plus end growth; and (iv) sequential subtraction analysis. The combination of these approaches allowed us to obtain data where the density of microtubules had previously prevented conventional methods to be applicable. In the steady state, nascent microtubules grew persistently from the centrosome towards the cell margin. Frequently, they arrived at the cell margin without undergoing any transition to the shortening phase. In contrast to the growth of microtubules, shortening of the plus ends from the periphery was non-persistent; that is, rescue was frequent and the extent of shortening showed a distribution of lengths reflecting a stochastic process. The combination of persistent growth and a cell boundary led to a difference in apparent microtubule behavior in the cell interior compared with that near the cell margin. Whereas microtubules in the cell interior showed asymmetric transition frequencies, their behavior near the cell margin showed frequent fluctuations between phases of shortening and growth. Complete microtubule turnover was accomplished by the relatively rare episodes of shortening back to the centrosome. Release from the centrosome with subsequent minus end shortening also occurred but was a minor mechanism for microtubule turnover compared with the plus end pathway. We propose a life cycle for a microtubule which consists of rapid growth from the centrosome to the cell margin followed by an indefinite period of fluctuations of phases of shortening and growth. We suggest that persistent growth and asymmetric transition frequencies serve the biological function of providing a mechanism by which microtubules may rapidly accommodate to the changing shape and advancing edge of motile cells. 

Blue light modulation of ion transport in the slime mutant of Neurospora crassa

Levina N.N., Dunina-Barkovskaya A.Y., Shabala S., Lew R.R. 

Journal of Membrane Biology 188 (2002) 213-222 

Blue light is the primary entrainment signal for a number of developmental and morphological processes in the lower eucaryote Neurospora crassa. Blue light regulates photoactivation of carotenoid synthesis, conidiation, phototropism of perithecia and circadian rhythms. Changes in the electrical properties of the plasma membrane are one of the fastest responses to blue light irradiation. To enable patch- clamp studies on light-induced ion channel activity, the wall- less slime mutant was used. Patch-clamp experiments were complemented by non-invasive ion-selective measurements of light-induced ion fluexes of slime cells using the vibrating probe technique. Blue light usually caused a decrease in conductance within 2-5 minutes at both negative and positive voltages, and a negative shift in the reversal potential in whole-cell patch-clamp measurements. Both K+ and Cl- channels contribute to the inward and outward currents, based on the effects of TEA (10 mm) and DIDS (500 ?m). However, the negative shift in the reversal potential indicates that under blue light the Cl- conductance becomes dominant in the electrical properties of the slime cells due to a decrease of K+ conductance. The ion-selective probe revealed that blue light induced the following changes in the net ion fluxes within 5 minutes: 1) decrease in H+ influx; 2) increase in K+ efflux; and 3) increase in Cl- influx. Ca2+ flux was unchanged. Therefore, blue light regulates an ensemble of transport processes: H+, Cl-, and K+ transport. 

Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain

Nikolaeva O.P., Orlov V.N., Bobkov A.A., Levitsky D.I.

European Journal of Biochemistry 269 (2002) 5678-5688

The thermal unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by differential scanning calorimetry. In the absence of nucleotides, trypsin splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This cleavage has no appreciable influence on the thermal unfolding of S1 examined in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the presence of F-actin. In the presence of ATP and in the complexes S1.ADP.Vi or S1.ADP.BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage strongly decreases the thermal stability of S1 by shifting the maximum of its thermal transition by about 70C to a lower temperature, from 500C to 42.40C, whereas the cleavage at both these sites causes dramatic destabilization of the S1 molecule leading to total loss of its thermal transition. Our results show that S1 with ATP-induced N-terminal cleavage is able, like uncleaved S1, to undergo global structural changes in forming the stable ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved by trypsin in the N-terminal region is unable, unlike S1, to undergo structural changes induced by interaction with F-actin that are expressed in a 4-50C shift of the S1 thermal transition to higher temperature. Thus, the cleavage between Arg23 and Ile24 does not significantly affect nucleotide-induced structural changes in the S1, but it prevents structural changes that occur when S1 is bound to F-actin. The results suggest that the N-terminal region of the S1 heavy chain plays an important role in structural stabilization of the entire motor domain of the myosin head, and a long-distance communication pathway may exist between this region and the actin-binding sites.

Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility 

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

Proc. Natl. Acad. Sci. USA 99 (2002) 10452-10457 

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior-posterior polarity is unknown. To dissect the roles of microtubule assembly and of Rho-dependent contractility on structural polarization of cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree of polarization by developing a highly elongate cell body with multiple narrow processes extended from the edges of the cell. Treatment of fibroblasts with nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid or polygonal cells having broad, flattened lamellae that did not form long lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not display anterior-posterior polarization. When treated with Y-27632, the cells acquired a polarized, elongate shape with narrow protrusions and wide lamellas. Nocodazole alone or in combination with Y-27632 did not change the discoid shape of epitheliocytes, however treatment with Y-27632 produced thinning of the lamellar cytoplasm. We conclude that microtubules provide the necessary framework for polarization of fibroblasts and epitheliocytes, whereas Rho-regulated contractility modulates the degree of polarization of fibroblasts and completely inhibits polarization in epitheliocytes. 

Responses of reticulospinal neurons in intact lamprey to pitch tilt

Pavlova E.L., Deliagina T.G. 

Journal of Neurophysiology 88 (2002) 1136-1146 

In the swimming lamprey, a postural control system maintains a definite orientation of the animal's longitudinal axis in relation to the horizon (pitch angle). Operation of this system is based on vestibular reflexes. Important elements of the postural network are the reticulospinal (RS) neurons, which are driven by vestibular input and transmit commands for postural corrections from the brain stem to the spinal cord. Here we describe responses to vestibular stimulation (rotation of the animal in the pitch plane) in RS neurons of intact lampreys. The activity of neurons was recorded from their axons in the spinal cord by chronically implanted arrays of macroelectrodes. From the multielectrode recordings of mass activity, discharges in individual axons were extracted by means of a spike-sorting program, and the axon position in the spinal cord and its conduction velocity were determined. Vestibular stimulation was performed by rotating the animal in steps of 45 degrees throughout 360 degrees or by periodical "trapezoid" tilts between the nose-up and -down positions. Typically, the RS neurons exhibited both dynamic responses (activity during movement) and static responses (activity in a new sustained position). The neurons were classified into two groups according to their pattern of response. Group UP neurons responded preferentially to nose-up rotation with maximal activity at 0-135 degrees up. Group DOWN neurons responded preferentially to nose-down rotation with maximal activity at 0-135 degrees down. Neurons of the two groups also differed in the position of their axons in the spinal cord and axonal conduction velocity. An increase in water temperature, which presumably causes a downward turn in swimming lampreys, affected the activity in the UP and DOWN groups differently, so that the ratio UP responses to DOWN responses increased. We suggest that the UP and DOWN groups mediate the opposing vestibular reflexes and cause the downward and upward turns of the animal, respectively. The lamprey will stabilize the orientation in the pitch plane at which the effects of UP and DOWN groups are equal to each other. In addition to the main test (rotation in the pitch plane), the animals were also tested by rotation in the transverse (roll) plane. It was found that 22% of RS neurons responding to pitch tilts also responded to roll tilts. The overlap between the pitch and roll populations suggests that the RS pathways are partly shared by the pitch and roll control systems.

Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro

Prass K., Ruscher K., Karsch M., Isaev N., Megow D., Priller J., Scharff A., Dirnagl U., Meisel A. 

Journal of Cerebral Blood Flow and Metabolism 22 (2002) 520-525 

The widely prescribed drug desferrioxamine is a known activator of the hypoxia-inducible transcription factor I (HIF-1) and the subsequent transcription of erythropoietin. In the brain. HIF-1 is a master switch of the transcriptional response to hypoxia, whereas erythropoietin is a potent neuroprotectant. The authors show that desferrioxamine dose-dependently and time-dependently induces tolerance against focal cerebral ischemia in rats and mice, and against oxygen-glucose deprivation in purified cortical neurons. Desferrioxamine induced HIF-1 DNA binding and transcription of erythropoietin in vivo, the temporal kinetics of which were congruent with tolerance induction. Desferrioxamine is a promising drug for the induction of tolerance in humans when ischemia can be anticipated. 

Isolation of the chromocenter fraction from mouse liver nuclei 

Prusov A.N., Zatsepina O.V. 

Biochemistry (Moscow) 67 (2002) 423-431 

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non- histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase. 

Sequencing of flagellin genes from Natrialba magadii provides new insight into evolutionary aspects of archaeal flagellins

Serganova I., Ksenzenko V., Serganov A., Meshcheryakova I., Pyatibratov M., Vakhrusheva O., Metlina A., Fedorov O.

Journal of Bacteriology 184 (2002) 318-322 

We have determined the nucleotide sequence of a flagellin gene locus from the haloalkaliphilic archaeon Natrialba magadii, identified the gene products among proteins forming flagella, and demonstrated cotranscription of the genes. Based on the sequence analysis we suggest that different regions of the genes might have distinct evolutionary histories including possible genetic exchange with bacterial flagellin genes.

Respiration and mitochondrial membrane potential are not required for apoptosis and anti-apoptotic action of Bcl-2 in HeLa cells 

Shchepina L.A., Popova E.N., Pletjushkina O.Y., Chernyak B.V. 

Biochemistry (Moscow) 67 (2002) 222-226 

The release of cytochrome c from intermembrane space of mitochondria into cytosol is one of the critical events in apoptotic cell death. The important anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it was shown that apoptosis and release of cytochrome c induced by staurosporine or by tumor necrosis factor-a in He La cells were not affected by inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers (CCCP, DIN P) that decrease the membrane potential at the inner mitochondrial membrane. The inhibitors of respiration and the uncouplers did not affect also the anti-apoptotic activity of Bcl-2. 

Immunocytochemical study of PCNA distribution in nuclei with stabilized and non-stabilized nuclear matrix 

Sheval E.V., Polyakov V.Y. 

Biologicheskie Membrany 19 (2002) 237-242 

Interchromatin compartment behaviour during cell permeabilization and extraction was analyzed in human cultured cells (HeLa). In cells permeabilized with the buffer which destabilized nuclear matrix the reduced staining (compare with nuclei with stabilized nuclear matrix) of interchromatin space by toluidine blue was observed. This suggested that a part of acidic proteins and/or RNA was extracted from the interchromatin material. Using antibodies to a protein of interchromatin material - PCNA, two population of this protein were observed. The replication factory variant was present in the nuclear matrix fraction, whereas the protein non- participated in replication and homogeneously distributed in nucleoplasm was absent from the nuclear matrix. The nucleoplasmic PCNA was extracted during permeabilization in the buffer, which destabilized nuclear matrix. Thus, the phenomenon of reduced staining by toluidine blue was, probably, due to extraction of non-matrix compounds of the interchromatin material. The data of present work suggest that proteins behaviour during nuclear matrix preparation do not depend exclusively on the preparation procedure, but in part on the protein state. 

Organization of higher-level chromatin structures (chromomere, chromonema and chromatin block) examined using visible light- induced chromatin photo-stabilization 

Sheval E.V., Prusov A.N., Kireev I.I., Fais D., Polyakov V.Y. 

Cell Biology International 26 (2002) 579-591 

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100 nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2 M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron- dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high- salt buffers from non-irradiated nuclei. 

Characteristics of nucleolar organization and function under condition of complete spatial in situ disassembly of its main structural components

Smirnova O.Iu., Dudnik O.A., Zatsepina O.V.

Tsitologiia 44 (2002) 5-13

It is well known that fibrillar centers (FC) constitute an essential structural component of the active nucleolus in mammalian cells, yet their role in regulation of ribosomal gene transcription still remains an open question. Here, we studied the activity of endogenous RNA polymerase I upon partial and complete unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of hypotonically-treated cells was shown to be essentially the same as in the control untreated cells. Moreover, the sites of BrUTP incorporation, which revealed the active PNA polymerase I, were completely coincident with UBF-binding sites. These observations allow to conclude that structural integrity of FCs is not a prerequisite for maintenance of the active RNA polymerase I transcriptional complex. When the action of hypotonic shock was ceased and the cells were transferred to a complete cultural medium, the swollen nucleoli recovered to the control state. Therefore it is possible to conclude that none of the main morphological nucleolar counterparts, such as FCs, dense fibrillar component or the pars granulosa, is responsible for the maintenance of the nucleolar structural and functional integrity. A suggestion is made that this role may be played by the nucleolar matrix associated with the RNA polymerase I transcriptional complex.

Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium

Tsiavaliaris G., Fujita-Becker S., Batra R., Levitsky D.I., Kull F.J., Geeves M.A., Manstein D.J.

EMBO Reports 3 (2002) 1099-105

Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants' affinity for actin in the presence of ATP.

An Analysis of Methyltransferase SsoII DNA Contacts in the Enzyme Substrate Complex 

Vorob'eva O.V., Karyagina A.S., Volkov E.M., Viryasov M.B., Oretskaya T.S., Kubareva E.A.

Russian Journal of Bioorganic Chemistry 28 (2002) 363-370

The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme-substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A ћ T by the G ћ C pair in the methylation site did not affect enzyme-DNA interaction, whereas the use of a substrate with one strand methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.


 

II. BIOENERGETICS AND PHOTOSYNTHESIS

A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect 

Abdullaev Z.K., Bodrova M.E., Chernyak B.V., Dolgikh D.A., Kluck R.M., Perverzev M.O., Arseniev A.S., Efremov R.G., Kirpichnikov M.P., Mokhova E.N., Newmeyer D.D., Roder H., Skulachev V.P. 

Biochemical Journal 362 (2002) 749-754 

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal a-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H2O2 production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other 'horse' modifications in the N-terminal a-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome e-mediated apoptosis, in contrast with the horse K72R. K72G. K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12. 

Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation 

Antonenko Y.N., Borisenko V., Melik-Nubarov N.S., Kotova E.A., Woolley G.A. 

Biophysical Journal 82 (2002) 1308-1318 

The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2- aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dinners in the membrane via clustering of channels. 

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization 

Azarkina N., Konstantinov A.A. 

Journal of Bacteriology 184 (2002) 5339-5347 

At a pH of less than or equal to 7, respiration of Bacillus subtilis cells on endogenous substrates shut down almost completely upon addition or an uncoupler (carbonyl cyanide m-chlorophenylhydrazone [CCCP]) and a K+-ionophore (valinomycin). The same effect was observed with cell spheroplasts lacking the cell wall. The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K+- valinomycin was below 100 nM. Either CCCP or valinomycin alone was much less efficient than the combination of the two. The inhibitory effect was easily reversible and depended specifically on the H+ and K+ concentrations in the medium. Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD+), which intercept reducing equivalents at the level of menaquinol. Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling. The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores. We conclude that the menaquinone-dependent electron transfer in the B. subtilis respiratory chain is facilitated, thermodynamically or kinetically, by membrane energization. A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant of B. subtilis lacking succinate:quinone reductase as well as for substrates other than succinate. Possible mechanisms of the energy-dependent regulation of menaquinone- dependent respiration in B. subtilis are discussed. 

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii 

Bertsova Y.V., Bogachev A.V. 

Biochemistry (Moscow) 67 (2002) 22-626 

A part of the gene encoding cbb3-type cytochrome oxidase CcoN Subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c4 and c5. Thus, the A. vinelandii respiratory chain is shown to contain cbb3-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations 

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

Bogachev A.V., Bertsova Y.V., Ruuge E.K., Wikstrom M., Verkhovsky M.I.

Biochim Biophys Acta 1556 (2002) 113-120 

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.

On the Involvement of the Water-Polaron Mechanism in Energy Trapping by Reaction Centers of Purple Bacteria

Borisov A.Yu., Kuznetsova S.A.

Biochemistry (Moscow) 67 (2002) 1224-1229

A locus for binding a mobile water molecule was searched for in the immediate vicinity of the special pair in the reaction center. Using the PROTEUS PC_program (a part of the GRASP package) atomic structures of the reaction centers were analyzed in purple bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides. In both structures the loci for binding mobile water molecules were found at the distance of about 4.5 A from the middle of the special pair in the reaction center. The reorientation of a hydrogen atom of this water molecule in the electric field of the excited special pair required energy of no less than 40 MeV that corresponded to predictions of the water_polarization model of trapping of electron excitation which was developed by M.V. Fok and one of the authors of this article.

Modeling of Energy Migration and Trapping in Purple Bacteria: Analysis of Limit Expressions

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

Biophysica 47 (2002) 272-283

Homogeneous pigment assemblies were considered, which are close in their characteristics to those in the purple bacteria Rhodospirillum rubrum and some others. Expressions were derived for the limit lifetime of electron excitations in these assemblies and for the limit quantum yield of trapping of excitations by reaction centers, all of which are in an active state. Mathematical modeling confirmed that these quantities depend on three parameters of molecular assemblies: the ratio between the number of core bacteriochlorophyll molecules and the number of reaction centers, the rate constant for trapping of electron excitations by reaction centers, and the rate constant for trivial deactivation of electron excitations in all molecules. It was shown that, with increasing energy migration rate constants, the lifetime of electron excitations and the quantum yield of trapping of electron excitations by reaction centers tend to their respective limit values. The closeness of the experimentally measured values of these quantities to the said limit values argues in favor of that the corresponding photosynthetic units are migration-limited and that the energy transfer from a molecular antenna to reaction centers is highly efficient.

Defects in mitochondrial respiratory complexes III and IV, and human pathologies 

Borisov V.B.

Molecular Aspects of Medicine 23 (2002) 385-412

Here, relationships between alterations in tissue-specific content, protein structure, activity, and/or assembly of respiratory complexes III and IV induced by mutations in corresponding genes and various human pathologies are reviewed. Cytochrome bc1 complex and cytochrome c oxidase (COX) deficiencies have been detected in a heterogeneous group of neuromuscular and non-neuromuscular diseases in childhood and adulthood, presenting a number of clinical phenotypes of variable severity. Such disorders can be caused by mutations located either in mitochondrial genes or in nuclear genes encoding structural subunits of the complexes or corresponding assembly factors/chaperones. Of the defects in mitochondrial DNA genes, mutations in cytochrome b subunit of complex III, and in structural subunits I-III of COX have been described to date. As to defects in nuclear DNA genes, mutations in genes encoding the complexes assembly factors such as the BCS1L protein for complex III; and SURF-1, SCO1, SCO2, and COX10 for complex IV have been identified so far.

Interactions between Heme d and Heme b595 in Quinol Oxidase bd from Escherichia coli: A Photoselection Study Using Femtosecond Spectroscopy 

Borisov V.B., Liebl U., Rappaport F., Martin J.L., Zhang J., Gennis R.B., Konstantinov A.A., Vos M.H.

Biochemistry 41 (2002) 1654-1662 

Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O2-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b595 on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b595 allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b595 transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b595, causing induction/loss of an absorption band centered at ~435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b595relative to the static spectrum. No evidence for transient binding of CO to heme b595 after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of <15 ps is found to be diminished more than 3-fold when heme b595 is oxidized rather than reduced. In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor 

Domnina L.V., Ivanova O.Y., Cherniak B.V., Skulachev V.P., Vasiliev J.M. 

Biochemistry (Moscow) 67 (2002) 37-746 

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor-a (TNF-a). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF-a to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria. 

Insulinotropic activity of the imidazoline derivative RX871024 in the diabetic GK rat 

Efanov A.M., Appelskog I.B., Abdel-Halim S.M., Khan A., Branstrom R., Larsson O., Ostenson C.G., Mest H.J., Berggren P.O., Efendic S., Zaitsev S.V.

American Journal of Physiol. Endocrinol. Metab. 282 (2002) E117-124 

The insulinotropic activity of the imidazoline derivative RX871024 was compared in pancreatic islets from nondiabetic Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats. RX871024 significantly stimulated insulin secretion in islets from both animal groups. The insulinotropic activity of RX871024 was higher than that of the sulfonylurea glibenclamide. This difference was more pronounced in islets from GK rats compared with Wistar rat islets. More importantly, RX871024 substantially improved glucose sensitivity in diabetic beta-cells, whereas glibenclamide stimulated insulin secretion about twofold over a broad range of glucose concentrations in nondiabetic and diabetic rats. RX871024 induced a faster increase in cytosolic free Ca2+ concentration and faster inhibition of ATP-dependent K+ channel activity in GK rat islets compared with Wistar rat islets. RX871024 also induced a more pronounced increase in diacylglycerol concentration in GK rat islets. These data support the idea that imidazoline compounds can form the basis for the development of novel drugs for treatment of type 2 diabetes, which can restore glucose sensitivity in diabetic beta-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 N+/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) 32-440 

The role of putative Na+/H+ antiporters encoded by nhaS1 (slr-1727), 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. 

Chromatophore vesicles of Rhodobacter capsulatus contain on average one FOF1-ATP synthase each 

Feniouk B.A., Cherepanov D.A., Voskoboynikova N.E., Mulkidjanian A.Y., Junge W. 

Biophysical Journal 82 (2002) 1115-1122 

ATP synthase is a unique rotary machine that uses the transmembrane electrochemical potential difference of proton D? over tilde +H to synthesize ATP from ADP and inorganic phosphate. Charge translocation by the enzyme can be most conveniently followed in chromatophores of phototrophic bacteria (vesicles derived from invaginations of the cytoplasmic membrane). Excitation of chromatophores by a short flash of light generates a step of the proton-motive force, and the charge transfer, which is coupled to ATP synthesis, can be spectrophotometrically monitored by electrochromic absorption transients of intrinsic carotenoids in the coupling membrane. We assessed the average number of functional enzyme molecules per chromatophore vesicle. Kinetic analysis of the electrochromic transients plus/minus specific ATP synthase inhibitors (efrapeptin and venturicidin) showed that the extent of the enzyme-related proton transfer dropped as a function of the inhibitor concentration, whereas the time constant of the proton transfer changed only marginally. Statistical analysis of the kinetic data revealed that the average number of proton- conducting F0F1-molecules per chromatophore was approximately one. Thereby chromatophores of Rhodobacter capsulatus provide a system where the coupling of proton transfer to ATP synthesis can be studied in a single enzyme/single vesicle mode. 

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from about half of chromatophores

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

Biochim. Biophys. Acta 1506 (2001) 189-203

F0F1-ATP synthase (H+-ATP synthase, F0F1) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (Pi). Structurally the enzyme consists of a membrane-embedded proton-translocating F0 portion and a protruding hydrophilic F1 part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F0F1 (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F1 (efrapeptin) and of F0 (venturicidin), we decomposed the kinetics of the total proton flow through F0F1 into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F0. Taking the coupled proton flow, we calculated the H+/ATP ratio; it was found to be 3.3+0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F0F1 dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and Pi led to an increase in the fraction of chromatophores with at least one de-coupled F0(F1). The disclosed fraction of chromatophores that lacked proton-conducting F0(F1) (approx. 40% of the total amount) remained constant upon these treatments.

MHYT, a new integral membrane sensor domain

Galperin M.Y., Gaidenko T.A., Mulkidjanian A.Y., Nakano M., Price C.W.

FEMS Microbiol Letters 205 (2001) 17-23 

MHYT, a new conserved protein domain with a likely signaling function, is described. This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098. In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins. In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein. A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.

Inositol hexakisphosphate promotes dynamin mediated endocytosis 

Hoy M., Efanov A.M., Bertorello A.M., Zaitsev S.V., Olsen H.L., Bokvist K., Leibiger B., Leibiger I.B., Zwiller J., Berggren P.O., Gromada J. 

Proc. Natl. Acad. Sci. USA 99 (2002) 6773-6777 

Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP6) is under metabolic control and serves as a signal in the pancreatic beta cell stimulus-secretion coupling by increasing Ca2+- channel activity and insulin exocytosis. We now show that InsP6 also promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of InsP6 depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphaticlylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP6 thus may have an essential integral role in membrane trafficking. 

Neuroprotective effects of the antifungal drug clotrimazole 

Isaev N.K., Stelmashook E.V., Dirnagl U., Andreeva N.A., Manuhova L., Vorobjev V.S., Sharonova I.N., Skrebitsky V.G., Victorov I.V., Katchanov J., Weih M., Zorov D.B. 

Neuroscience 113 (2002) 47-53 

Pretreatment with 10 ?M of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 ?M clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca2+ overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca2+ overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment Using tetra-methylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse. 

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn2+ decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation 

Klishin S.S., Junge W., Mulkidjanian A.Y. 

Biochim. Biophys. Acta 1553 (2002) 177-182 

The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 (complex bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme bh (in the absence of Zn2+ this reaction vas masked by the fast re- oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c1 reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ < 100 ?M the reduction of heme bh remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme bh is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme bh oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex. 

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically 'dark') S1 state of carotenoids 

Krikunova M., Kummrow A., Voigt B., Rini M., Lokstein H., Moskalenko A., Scheer H., Razjivin A., Leupold D.

FEBS Letters 528 (2002) 227-229

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300-1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2Ag- state of rhodopin is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid-depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.


Kinetically different populations of O-pyromellityl-gramicidin channels induced by poly-L-lysines in lipid bilayers

Krylov A.V., Rokitskaya T.I., Kotova E.A., Yaroslavov A.A., Antonenko Y.N.

Journal of Membrane Biology 189 (2002) 119-130 

Clustering of membrane proteins, in particular of ion channels, plays an important role in their functioning. To further elucidate the mechanism of such ion channel activity regulation, we performed experiments with a model system comprising the negatively-charged gramicidin analog, O- pyromellitylgramicidin (OPg) that forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect of polylysines on the kinetics of OPg channels in BLM was studied by the method of sensitized photoinactivation. As found in our previous work, the interaction of polylysine with OPg led to the deceleration of the OPg photoinactivation kinetics, i.e., to the increase in the characteristic time of OPg photoinactivation. It was shown here that in a certain range of polylysine concentrations the photoinactivation kinetics displayed systematic deviations from a monoexponential curve and was well described by a sum of two exponentials. The deviations from the monoexponential approximation were more pronounced with polylysines having a lower degree of polymerization. These deviations increased also upon the elevation of the ionic strength of the bathing solution and the addition of calcium ions. A theoretical model is presented that relates the OPg photoinactivation kinetics at different concentration ratios of OPg and polylysine to the distribution of OPg molecules among OPg-polylysine clusters of different stoichiometry. This model is shown to explain qualitatively the experimental results, although the quantitative description of the whole body of evidence requires further development, assuming that the interaction of polylysine with OPg causes segregation of membrane domains enriched in OPg channels. The single-channel data, which revealed the insensitivity of the single-channel lifetime of OPg to the addition of polylysine, are in good agreement with the theoretical model. 

Ca2+-binding site in Rhodobacter sphaeroides cytochrome c oxidase 

Lee A., Kirichenko A., Vygodina T., Siletsky S.A., Das T.K., Rousseau D.L., Gennis R., Konstantinov A.A. 

Biochemistry 41 (2002) 8886-8898 

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca2+ ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca2+ reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca2+ coordination sphere in subunit 1, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca2+; their spectra are not perturbed by added Call or EGTA. The D485A mutant is active, binds to Ca2+ reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca2+ binding for both reduced and oxidized states of heme a. The K-d value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K-d = 1-3 ?M). The rate of Ca2+ binding with the D485A oxidase (kon = 5 x 103 M-1 s-1) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (similar to10-3 s-1) and highly temperature-dependent. The koff/kon ratio (190 nM) is about 30-fold higher than the equilibrium K-d of 6 nM, indicating, that formation of the Ca2+-adduct may involve more than one step. Sodium ions reverse the Ca2+-induced red shift of heme a and dramatically decrease the rate of Ca2+ binding to the D485A mutant COX. With the D485A mutant, 1 Ca2+ competes with 1 Na+ for the binding site, whereas 2 Na+compete with 1 Ca2+ for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na+binding site in bovine COX. No effect of Ca2+ binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Call plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function. 

Effect of synthetic Amphiphilic polyanions on the ion permeation through planar bilayer lipid membrane 

Maltseva E.A., Antonenko Y.N., Melik-Nubarov N.S., Yaguzhinsky L.S. 

Biologicheskie Membrany 19 (2002) 347-350 

The effects of synthetic amphipathic polyanions (Konig polyanion and a copolymer of polyacrylic acid with sterylacrylate) on the carrier-mediated ion transport across planar bilayer lipid membranes (BLM) have been studied. The binding of these polymers to the membrane increased the cationic current (complex of K+-valinomycin) and decreased the anionic current (TTFB-) across the membrane. Hydrophylic polyanions lacking large hydrophobic fragments did not affect the ionic currents across the BLM. It is concluded that the effect of polyanions on the carrier-induced ionic transport is mediated by the binding of hydrophobic fragments of polyanions to the BLM. This leads to the change in the BLM cur-rent due to the appearance of negative surface charge. The mechanism of the action of Konig polyanion on the transport systems of mitochondria is discussed. 

Analysis of effects of 2,2 ',5,5 '-tetrachlorobiphenyl on the flux control in oxidative phosphorylation system in rat liver mitochondria 

Mildaziene V., Nauciene Z., Baniene R., Demin O., Krab K.

Molecular Biology Reports 29 (2002) 35-40 

Modular kinetic analysis reveals that the environmental pollutant 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) affects a large number of steps in oxidative phosphorylation in rat liver mitochondria. 2,2',5,5'-TCB increases membrane permeability to ions, and inhibits NADH dehydrogenase, cytochrome bc1, cytochrome oxidase (all in the respiratory chain) and ATP-synthase (in the phosphorylation subsystem). Surprisingly, flux control distribution does not change. A kinetic model for oxidative phosphorylation was used to simulate these findings, and it was found that combined large changes in the processes indicated indeed left the flux control largely unchanged. In addition, computational analysis with the model indicated that the adenine nucleotide translocator might be inhibited by 2,2',5,5'-TCB. 

Temperature dependence of the epidermal growth factor receptor signaling network can be accounted for by a kinetic model

Moehren G., Markevich N., Demin O., Kiyatkin A., Goryanin I., Hoek J.B., Kholodenko B.N.

Biochemistry 41 (2002) 306-320 

Stimulation of isolated hepatocytes with epidermal growth factor (EGF) causes rapid tyrosine phosphorylation of the EGF receptor (EGFR) and adapter/target proteins, which was monitored with 1 and 2 s resolution at 37, 20, and 4 degrees C. The temporal responses detected for multiple signaling proteins involve both transient and sustained phosphorylation patterns, which change dramatically at low temperatures. To account quantitatively for complex responses, we employed a mechanistic kinetic model of the EGFR pathway, formulated in molecular terms as cascades of protein interactions and phosphorylation and dephosphorylation reactions. Assuming differential temperature dependencies for different reaction groups, such as SH2 and PTB domain-mediated interactions, the EGFR kinase, and the phosphatases, good quantitative agreement was obtained between computer-simulated and measured responses. The kinetic model demonstrates that, for each protein-protein interaction, the dissociation rate constant, koff, strongly decreases at low temperatures, whereas this decline may or may not be accompanied by a large decrease in the kon value. Temperature-induced changes in the maximal activities of the reactions catalyzed by the EGFR kinase were moderate, compared to such changes in the Vmax of the phosphatases. However, strong changes in both the Vmax and Km for phosphatases resulted in moderate changes in the Vmax/Km ratio, comparable to the corresponding changes in EGFR kinase activity, with a single exception for the receptor phosphatase at 4 degrees C. The model suggests a significant decrease in the rates of the EGF receptor dimerization and its dephosphorylation at 4 degrees C, which can be related to the phase transition in the membrane lipids. A combination of high-resolution experimental monitoring and molecular level kinetic modeling made it possible to quantitatively account for the temperature dependence of the integrative signaling responses.

Effect of Growth Conditions on the Synthesis of Terminal Oxidases in Methylobacillus flagellatus KT 

Muntyan M.S, Dinarieva T.Yu., Baev M.V., Netrusov A.I.

Archives of Biochemistry and Biophysics 398 (2002) 118-124 

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The a-region and especially the a-peaks at 568 and 573 nm and the a-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h-1 under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 mM under the constant dilution rate (m = 0.55 h-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (m = 0.55 h-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (d-mH+). 

Molecular identification of alkaliphilic and halotolerant strain Bacillus sp FTU as Bacillus pseudofirmus FTU 

Muntyan M.S., Tourova T.P., Lysenko A.M., Kolganova T.V., Fritze D., Skulachev V.P. 

Extremophiles 6 (2002) 195-199 

The systematic position of the alkaliphilic and halotolerant strain Bacillus sp. FTU was refined in view of the comprehensive taxonomic revision of the group of alkaliphilic and alkalitolerant Bacillus strains. Sequence analysis of almost the entire 16S rRNA gene of Bacillus sp. FTU revealed 99.8% homology with two Bacillus pseudofirmus strains. Subsequent DNA-DNA hybridization analysis confirmed the close relationship of Bacillus sp. FTU with the type strain of B. pseudofirmus (the level of homology reached 86%). Results of physiological and biochemical characterizations relevant for the group clearly underlined the positioning of strain FTU within this species. It is therefore concluded that Bacillus sp. FTU represents a strain of the alkaliphilic species B. pseudofirmus and is to be renamed as B. pseudofirmus FTU. The phylogeny of different Bacillus species is discussed using N- terminal sequence homologies of some caa(3)-type oxidase subunits. 

Exciton-vibrational relaxation and transient absorption dynamics in LH1 of Rhodopseudomonas viridis: A Redfield theory approach 

Novoderezhkin V., van Grondelle R. 

Journal of Physical Chemistry B 106 (2002) 6025-6037 

We explain the ultrafast transient absorption (TA) dynamics in the core LH1 antenna of Rhodopseudomonas viridis at 77 K (Monshouwer, R. Baltuska. A.: van Mourik, F.; van Grondelle, R. J, Phys. Chem. A 1998, 102, 4360) using a disordered exciton model with strong coupling to two vibrational modes and weak coupling of vibrational and electronic coordinates to the thermal bath. A quantitative fit of the excitation wavelength- dependent TA dynamics was obtained using the density matrix equation with the Redfield relaxation operator. The sequential and coherent contributions to the TA dynamics were analyzed separately. The time-dependent red shift of TA was explained in terms of exciton relaxation. The lifetimes of higher exciton states of 12-75 fs (depending on the state) were determined from the fit. 

Thyroxine reversibly inhibits the uncoupling action of protonophores on energy production in rat thymus lymphocytes 

Palamarchuk L.A., Mansurova S.E., Starkov A.A. 

Biochemistry (Moscow) 67 (2002) 468-472 

Earlier we reported that some thyroid and steroid hormones and also 6-ketocholestanol used in micromolar concentrations modulated the effects of protonophoric uncouplers on isolated mitochondria (Starkov et al, (1997) Biochim. Biophys. Acta, 1318, 173-183). In the present study we investigated the effects of a thyroid hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and mitochondria isolated from these cells. The resting (oligomycin-inhibited) respiration of the isolated intact lymphocytes was stimulated by the addition of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of micromolar concentrations of thyroxin decreased the rate of uncoupler-stimulated respiration and partially reversed uncoupler-induced decrease of membrane potential (DY). In experiments with mitochondria isolated from thymus lymphocytes the re-coupling effect of thyroxine was not observed. In this case thyroxine did not influence mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation of respiration and DY decrease induced with another uncoupler, SF6847. The data are discussed in terms of a hypothesis that aromatic uncouplers are transported into the cell by the thyroxine carrier of the plasma membrane. 

Effects of cold exposure in vivo and uncouplers and recouplers in vitro on potato tuber mitochondria 

Popov V.N., Markova O.V., Mokhova E.N., Skulachev V.P. 

Biochim. Biophys. Acta 1553 (2002) 232-237 

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate. nucleotides, and BSA in vitro on potato tuber mitochondria ha e been studied. Cold exposure of tubers Cor 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDR carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP. and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides ere absent when the nucleotides were added after carboxyatractlate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter, As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides. 

Membrane Dipole Potential Modulates Proton Conductance through Gramicidin Channel: Movement of Negative Ionic Defects inside the Channel 

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

Biophysical Journal 82 (2002) 865-873 

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H+ concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.

The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide

Rosen G.M., Tsai P., Weaver J., Porasuphatana S., Roman L.J., Starkov A.A., Fiskum G., Pou S.

Journal of Biological Chemistry 277 (2002) 40275-40280 

Tetrahydrobiopterin (H4B) is a critical element in the nitric-oxide synthase (NOS) metabolism of l-arginine to l-citrulline and NO.. It has been hypothesized that in the absence of or under nonsaturating levels of L-arginine where O2 reduction is the primary outcome of NOS activation, H4B promotes the generation of H2O2 at the expense of O2-.. The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H4B-bound NOS I and H4B-free NOS I, were used. Initial rates of NADPH turnover and O2 utilization were found to be considerably greater in the H4B-bound NOS I preparation than in the H4B-free NOS I preparation. In contrast, the initial generation of O2-. from the H4B-free NOS I preparation was found to be substantially greater than that measured using the H4B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O2-., we found that the initial rate of H2O2 production by H4B-bound NOS I was considerably greater than that for H4B-free NOS I.

Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model

Ruscher K., Freyer D., Karsch M., Isaev N., Megow D., Sawitzki B., Priller J., Dirnagl U., Meisel A.

Journal of Neuroscience 22 (2002) 10291-10301 

In an in vitro model of cerebral ischemia (oxygen glucose deprivation, OGD) we investigated whether erythropoietin (EPO) plays a critical role in ischemic preconditioning. We found that EPO time and dose-dependently induced protection against OGD in rat primary cortical neurons. Protection was significant at 5 min and reached a maximum at 48 hr after EPO application. Protection was blocked by the coapplication of a soluble Epo receptor (sEpoR) or an antibody against EpoR (anti-EpoR). Medium transfer from OGD-treated astrocytes to untreated neurons induced protection against OGD in neurons, which was attenuated strongly by the application of sEpoR and anti-EpoR. In contrast, medium transfer from OGD-treated neurons to untreated neurons induced protection against OGD that did not involve EPO. In astrocytes the OGD enhanced the nuclear translocation of hypoxia-inducible factor 1 (HIF-1), the major transcription factor regulating EPO expression. Consequently, transcription of EPO-mRNA was increased in astrocytes after OGD. Cultured neurons express EpoR, and the Janus kinase-2 (JAK-2) inhibitor AG490 abolished EPO-induced tolerance against OGD. Furthermore, EPO-induced neuroprotection as well as phosphorylation of the proapoptotic Bcl family member Bad was reduced by the phosphoinositide-3 kinase (PI3K) inhibitor LY294002. The results suggest that astrocytes challenged with OGD provide paracrine protective signals to neurons. We provide evidence for the following signaling cascade: HIF-1 is activated rapidly by hypoxia in astrocytes. After HIF-1 activation the astrocytes express and release EPO. EPO activates the neuronal EPO receptor and, subsequently, JAK-2 and thereby PI3K. PI3K deactivates BAD via Akt-mediated phosphorylation and thus may inhibit hypoxia-induced apoptosis in neurons. Our results establish EPO as an important paracrine neuroprotective mediator of ischemic preconditioning.

Induction of apoptosis in rat myocardium under anoxic conditions 

Saprunova V.B., Kazimirchuk S.A., Tonshin A.A., Bakeeva L.E., Yagujinsky L.S. 

Biochemistry (Moscow) 67 (2002) 246-253 

The effect of anoxic incubation of small slices of isolated rat hearts on respiration, internucleosomal DNA fragmentation, and mitochondrial ultrastructure was investigated. Anoxic incubation for 72 h induced apoptosis accompanied by internucleosomal DNA fragmentation and changes in respiration and mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In a significant part of the mitochondrial population there were signs of mitochondrial swelling and appearance of electron- dense mitochondria. Anoxia also induced the appearance of an atypical (and previously unknown) population of small electron- dense mitochondria. They were characterized by unusual localization inside electron-light mitochondria. Under anoxic conditions the inner mitochondrial membrane formed electron- dense ordered structures. All changes described here reflect two opposing processes occurring in mitochondria: apoptotic destruction and compensatory processes responsible for maintenance of mitochondria. 

Transfer of cationic antibacterial agents berberine, palmatine, and benzalkonium through bimolecular planar phospholipid film and Staphylococcus aureus membrane 

Severina I.I., Muntyan M.S., Lewis K., Skulachev V.P.

IUBMB Life 52 (2001) 321-324

Some organic cations are known to be electrophoretically imported into bacterial cells and actively extruded from these cells by multidrug resistance (MDR) pumps. We have studied penetration of plant antimicrobial agents berberine and palmatine and synthetic antiseptic benzalkonium chloride through black planar phospholipid membrane (BLM) and membrane of Staphylococcus aureus cells. Gradients of these cations across BLM generated an electric potential difference. Penetrating anion tetraphenyl borate and phloretin (a plant substance decreasing membrane dipole potential) stimulated this effect. Under optimal conditions, the magnitude of the electric potential was close to theoretical, that is, 60 mV/10-fold cation gradient. Berberine accumulated in S. aureus cells as shown by direct measurement of berberine with a berberine-sensitive electrode. The berberine accumulation was prevented by protonophore CCCP and was stimulated by mutation in the MDR pump NorA. It is concluded that the plant alkaloids and benzalkonium are penetrating cations and substrates of an MDR pump.

Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis 

Shchepina L.A., Pletjushkina O.Y., Avetisyan A.V., Bakeeva L.E., Fetisova E.K., Izyumov D.S., Saprunova V.B., Vyssokikh M.Y., Chernyak B.V., Skulachev V.P.

Oncogene 21 (2002) 8149-8157

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor a (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+-ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested.

Glucose metabolites inhibit protein phosphatases and directly promote insulin exocytosis in pancreatic beta-cells

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

Endocrinology 143 (2002) 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 beta-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 beta-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 beta-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 beta-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 beta-cell.

Programmed death phenomena: from organelle to organism 

Skulachev V.P.

Ann. N. Y. Acad. Sci. 959 (2002) 214-237

Programmed death phenomena appear to be inherent not only in living cells (apoptosis), but also in subcellular organelles (e.g., self-elimination of mitochondria, called mitoptosis), organs (organoptosis), and even whole organisms (phenoptosis). In all these cases, the "Samurai law of biology"--it is better to die than to be wrong--seems to be operative. The operation of this law helps complicated living systems avoid the risk of ruin when a system of lower hierarchic position makes a significant mistake. Thus, mitoptosis purifies a cell from damaged and hence unwanted mitochondria; apoptosis purifies a tissue from unwanted cells; and phenoptosis purifies a community from unwanted individuals. Defense against reactive oxygen species (ROS) is probably one of the primary evolutionary functions of programmed death mechanisms. So far, it seems that ROS play a key role in the mito-, apo-, organo-, and phenoptoses, which is consistent with Harman's theory of aging. Here a concept is described that tries to unite Weismann's hypothesis of aging as an adaptive programmed death mechanism and the generally accepted alternative point of view that considers aging as an inevitable result of accumulation in an organism of occasional injuries. It is suggested that injury accumulation is monitored by a system(s) actuating a phenoptotic death program when the number of injuries reaches some critical level. The system(s) in question are organized in such a way that the lethal case appears to be a result of phenoptosis long before the occasional injuries make impossible the functioning of the organism. It is stressed that for humans these cruel regulations look like an atavism that, if overcome, might dramatically prolong the human life span.

Programmed death in yeast as adaptation?

Skulachev V.P.

FEBS Letters 528 (2002) 23-26

During recent years, several pieces of indirect evidence of a programmed death in yeast have been published. Among them there are observations that some mammalian pro- or anti-apoptotic proteins induce or prevent the death of yeast; some toxic compounds kill yeast at lower concentrations if protein synthesis is operative; this death, as well as the death due to certain mutations, shows some apoptotic markers. In April 2002, the yeast programmed death concept received direct support. Madeo et al. [Madeo et al., Mol. Cell 9 (2002) 911-917] disclosed a caspase which is activated by H2O2 or aging and is required for the protein-synthesis-dependent death of yeast. Thus, a specific apoptosis-mediating protein was identified for the first time in Saccharomyces cerevisiae. Independently, Severin and Hyman [Severin, F.F., Hyman, A.A., Curr. Biol. 12 (2002) R233-R235] discovered that death of yeast, induced by a high level of a pheromone, is programmed. In particular, the death was found to be prevented by cycloheximide and cyclosporin A. It required mitochondrial DNA, cytochrome c and the pheromone-initiated protein kinase cascade. When haploids of opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone. Inhibition of mating proved to be favorable for death. Thus, pheromone not only activates mating but also eliminates yeast cells failing to mate. Such an effect should (i) stimulate switch of the yeast population from vegetative to sexual reproduction, and (ii) shorten the life span and, hence, accelerate changing of generations. As a result, the probability of appearance of new traits could be enhanced when ambient conditions turned for the worse.

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Starkov A.A., Polster B.M., Fiskum G.

Journal of Neurochemistry 83 (2002) 220-228 

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.

Structural determinants of fluorochemical-induced mitochondrial dysfunction

Starkov A.A., Wallace K.B.

Toxicol. Sci. 66 (2002) 244-252 

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.

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.M., Brdiczka D.

Molecular Biology Reports 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 Triton-X100 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-DeltaC 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-DeltaC 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. 

Steady-state spectroscopy of zinc-bacteriopheophytin containing LH1 - an in vitro and in silico study 

Wendling M., Lapouge K., van Mourik F., Novoderezhkin V., Robert B., van Grondelle R. 

Chemical Physics 275 (2002) 31-45 

By reversible dissociation of the light-harvesting complex 1 (LH1) of Rhodospirillum rubrum it is possible to (partly) exchange the bacteriochlorophyll (BChl) a by zinc- bacteriopheophytin (Zn-BPheo). After reassociation of the protein a complex is formed which can have different percentages of Zn-BPheo bound to the polypeptides [Biochemistry 39 (2000) 1091]. Low-temperature absorption spectra show a shift of the absorption maximum from 886 to 863 nm, when the native LH1 complex is compared to a modified complex containing 90% Zn-BPheo, whereas the positions of the absorption maxima of BChl a and Zn-BPheo differ by only 6 nm, when the pigments are bound to the isolated polypeptides. Using an exciton model with static disorder of site energies based on the ring-like structure of LH1 we can describe this shift by assuming a difference in the excitonic coupling originating solely from the different dipole strength of the exchanged Zn-BPheo compared with the original BChl a. We estimate that in LH1 the nearest-neighbour interaction energy of two BChl a molecules is around 400 cm-1 and the diagonal disorder is around 600 cm-1. Furthermore, we determined if the energy transfer in pigment- modified complexes is similar to native LH1. This can be observed by selectively exciting electronic states depending on their energy and measuring the polarized emission spectra at low temperature. The native complex was compared to a complex containing 70% Zn-BPheo. The fluorescence anisotropy and the shift of the emission maximum in native LH1 and 70%-Zn-BPheo- LH1 depending on the excitation wavelength can generally be described within the same disordered exciton model, extended in a simple way with line shapes. The model proved to be simple and robust when applied to these engineered light-harvesting complexes. 

Nuclear wave packet motion between P* and P+BA- potential surfaces with a subsequent electron transfer to HA in bacterial reaction centers at 90 K. Electron transfer pathway 

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

Biochemistry 41 (2002) 14019-14027

In Rhodobacter sphaeroides R-26 reaction centers (RCs) the nuclear wave packet induced by 25 fs excitation at 90 K moves on the primary electron donor P* potential energy hypersurface with initial frequency at approximately 130 cm-1 (monitored by stimulated emission measurement). At the long-wavelength side of P* stimulated emission at 935 nm the wave packet is transferred to the surface with P+BA- character at 120, 380, 1.2 fs, etc. delays (monitored by measurement of the primary electron acceptor P+BA- band at 1020 nm). However, only beginning from 380 fs delay and later the relative stabilization of the state P+BA- is observed. This is accompanied by the electron transfer to bacteriopheophytin HA (monitored by HA band measurement at 760 nm). The most active mode of 32 cm-1 in the electron transfer and its overtones up to the seventh were found in the Fourier transform spectrum of the oscillatory part of the kinetics of the P* stimulated emission and of the P+BA- and P+BA- formation. This mode and its overtones are apparently populated via the 130 cm-1 vibrational mode. The deuteration of the sample shifts the fundamental frequency (32 cm-1) and all overtones by the same factor of approximately 1.3. This mode and its overtones are suppressed by a factor of approximately 4.7 in the dry film of RCs. The results obtained indicate that the 32 cm-1 mode might be related to a rotation of hydrogen-containing groups (possibly the water molecule) participating in the modulation of the primary electron transfer from P* to BA- in at least 35% of RCs. The Brookhaven Protein Data Bank (1PRC) displays the water molecule located at the position HOH302 between His M200 (axial ligand for PB) and the oxygen of ring V of BA which might be a part (approximately 35%) of the molecular pathway for electron transfer from P* to BA.

Nuclear wavepacket motion between P* and P+BA- potential surfaces with subsequent electron transfer to HA in bacterial reaction centers. 1. Room temperature 

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

Biochemistry 41 (2002) 2667-2674 

Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P* and of the charge transfer states P+BA- and P+HA- were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where BA and HA are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P* at 935 nm, of the absorption band of BA- at 1020 nm, and of the bleaching band of HA at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm-1 surface of P* is directly induced by light absorption in P. When the wavepacket approaches the intersection between P* and P+BA- surfaces at 120 and 380 fs delays, the formation of intermediate mixed- state emitting light at 935 nm (P*) and absorbing light at 1020 nm (P+BA-) takes place. At the latter time, the wavepacket is transferred to the 32 cm-1 mode which can belong to the P* hypersurface effectively transferring the wavepacket to the P+BA- surface or can represent adiabatic surface which is formed by the states P* and P+BA-. The wavepacket motion on the P+BA- surface or on the P+BA- part of the mixing surface is accompanied by irreversible electron transfer to HA. This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm-1 mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm-1 to the 32 cm-1 mode is discussed. 

Light control over the size of an antenna unit building block as an efficient strategy for light harvesting in photosynthesis 

Yakovlev A.G., Taisova A.S., Fetisova Z.G. 

FEBS Letters 512 (2002) 129-132 

It was shown that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon lowering growth light intensities led to enhancement of the hyperchromism of the BChl c Qy absorption band of the green photosynthetic bacterium Chloroflexus aurantiacus. With feratosecond difference absorption spectroscopy, it was shown that the amplitude of bleaching of the oligomeric BChl c Qy band (as compared to that for monomeric BChl a) increased with increasing BChl c content in chlorosomes. This BChl c bleaching amplitude was about doubled as the chlorosomal antenna size was about trebled. Both sets of findings clearly show that a unit BChl c aggregate in the chlorosomal antenna is variable in size and governed by the grow light intensity, thus ensuring the high efficiency of energy transfer within the BChl c antenna regardless of its size. 

Tuning the membrane surface potential for efficient toxin import 

Zakharov S.D., Rokitskaya T.I., Shapovalov V.L., Antonenko Y.N., Cramer W.A. 

Proc Natl Acad Sci USA 99 (2002) 8654-8659 

Membrane surface electrostatic interactions impose structural constraints on imported proteins. An unprecedented sensitive dependence on these constraints was seen in the voltage-gated Import and channel formation by the C-terminal pore-forming domain of the bacteriocin, colicin El. At physiological ionic strengths, significant channel current was observed only in a narrow interval of anionic lipid content ([L-1]), with the maximum current (I-max) at 25-30 mol% (dioleoyl)- phosphatidylglycerol ([L-1]max) corresponding to a surface potential of the lipid bilayer in the absence of protein, Y(0)max = -60 + 5 mV. Higher ionic strength shifted [L-1]max to larger values, but Y(0)max remained approximately constant. It is proposed that the channel current (i) increases and (ii) decreases at Y(0) values <55 mV and >65 mV, because of (i) electrostatic Interactions needed for effective insertion of the channel polypeptide and (ii) constraints due to electrostatic forces on the flexibility needed for cooperative insertion into the membrane. The loss of flexibility for Y(0) much greater than 65 mV was demonstrated by the absence of thermally induced intraprotein distance changes of the bound polypeptide. The anionic lipid content, 25-30 mol%, corresponding to the channel current maxima, is similar to that of the target Escherichia coli cytoplasmic membrane and membranes of mesophilic microorganisms. This suggests that one reason the membrane surface potential is tuned in vivo is to facilitate protein import. 

 

 

III. MATHEMATICAL MODELS IN BIOLOGY

 

 

Adaptive algorithm of automated annotation 

Leontovich A.M., Brodsky L.I., Drachev V.A., Nikolaev V.K. 

Bioinformatics 18 (2002) 838-846 

Motivation: It is common knowledge that the avalanche of data arriving from the sequencing projects cannot be annotated either experimentally or manually by experts. The need for a reliable and convenient tool for automated sequence annotation is broadly recognized. Results: Here, we describe the Adaptive Algorithm of Automated Annotation (A4) based on a statistical approach to this problem. The mathematical model relates a set of homologous sequences and descriptions of their functional properties, and calculates the probabilities of transferring a sequence description onto its homologue. The proposed model is adaptive, its parameters (distribution characteristics, transference probabilities, thresholds, etc.) are dynamic, i.e. are generated individually for the sequences and various functional properties (words of the description), The proposed technique significantly outperforms the widely used test for frequency threshold, which is a special case of our model realized for the simplest set of parameters. The prediction technique has been realized as a computer program and tested on a random sequence sampling from SWISS-PROT. 

 

IV. MOLECULAR VIROLOGY


 
 

Genome Instability in Picornaviruses 

Agol V.I.

Molecular Biology (Moscow) 36 (2002) 216-222

Picornaviruses are small animal RNA viruses and include etiological agents of poliomyelitis, foot and mouth disease, hepatitis A, etc. Replication of their genome results in many mutations, which are close in number to a viability threshold. Hence every virus population contains a great variety of genomes and represents a quasispecies. Covalent rearrangements (deletions, insertions, recombination) also contribute to genome variation and arise by replicative and nonreplicative mechanisms, which are still poorly understood. Only a minor fraction of all new changes is fixed during evolution. The fixation is based on two principally different ways of selection: with (positive and negative selection) and without (random selection of nonrepresentative variants) regard to the phenotype. In natural evolution of picornaviruses, the latter way is prevalent, and most fixed mutations are phenotypically neutral. To understand the mechanisms of evolution, it is necessary to evaluate the biological significance of particular genetic changes. Several new approaches to this problem have recently been proposed.

Viral Infection and Cell Differentiation 

Agol V.I.

Russian Journal of Developmental Biology 33 (2002) 279-283

The dependence of viral reproduction and success of viral infection on cell differentiation is briefly reviewed at the example of picornaviruses-small RNA viruses of animals. In particular, the role of the cell factors in viral tropism, control of viral RNA translation, and the pattern of infected cell death are discussed. 

Long-term circulation of vaccine-derived poliovirus that causes paralytic disease 

Cherkasova E.A., Korotkova E.A., Yakovenko M.L., Ivanova O.E., Eremeeva T.P., Chumakov K.M., Agol V.I. 

Journal of Virology 76 (2002) 6791-6799 

Successful implementation of the global poliomyelitis eradication program raises the problem of vaccination against poliomyelitis in the posteradication era. One of the options under consideration envisions completely stopping worldwide the use of the Sabin vaccine. This strategy is based on the assumption that the natural circulation of attenuated strains and their derivatives is strictly limited. Here, we report the characterization of a highly evolved derivative of the Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a 7-month-old immunocompetent baby in an apparently adequately immunized population. Analysis of the genome of this isolate showed that it is a double (type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations accumulated in both the type 1-derived and type 2-derived portions of the recombinant genome suggests that both had diverged from their vaccine predecessors similar to 2 years before the onset of the illness. This fact, along with other recent observations, points to the possibility of long-term circulation of Sabin vaccine strain derivatives associated with an increase in their neurovirulence. Comparison of genomic sequences of this and other evolved vaccine-derived isolates reveals some general features of natural poliovirus evolution. They include a very high preponderance and nonrandom distribution of synonymous substitutions, conservation of secondary structures of important cis-acting elements of the genome, and an apparently adaptive character of most of the amino acid mutations, with only a few of them occurring in the antigenic determinants. Another interesting feature is a frequent occurrence of tripartite intertypic recombinants with either type 1 or type 3 homotypic genomic ends. 

Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry 

Dorokhov Y.L., Skulachev M.V., Ivanov P.A., Zvereva S.D., Tjulkina L.G., Merits A., Gleba Y.Y., Hohn T., Atabekov J.G. 

Proc. Natl. Acad. Sci. USA 99 (2002) 5301-5306 

The internal ribosome entry sites (IRES), IRESCP,148CR IRESMP,75,(CR) and IRE MP, precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus- encephalomyocarditis virus-in plant, yeast, and HeLa cells identified the 148-nt IRESCP,148CR as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRESCP,148CR are responsible for these features. On the basis oil' those findings, we designed artificial PARS-containing elements and showed that they, too, promote internal translation from dicistronic transcripts in vitro, in tobacco protoplasts and in HeLa cells. The maximum IRES activity was obtained from multiple copies of either A4GA2G2 or GA2-5 SCR as contained in IRESCP,148CR. Remarkably, even homopolymeric poly(A) was moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a database search for existing PARS sequences in T-untranslated regions (5'UTR) of genes in tobacco genome allowed the easy identification of a number of IRES candidates, in particular in the 5'UTR of the gene encoding Nicotiana tabacum heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5'UTR of NtHSF1 turned out to be an IRES element active in vitro, in plant protoplasts and HeLa cells. We predict that PARS elements, when found in other mRNAs, will show a similar activity. 

RNA helicase activity of the plant virus movement proteins encoded by the first gene of the triple gene block 

Kalinina N.O., Rakitina D.V., Solovyev A.G., Schiemann J., Morozov S.Y. 

Virology 296 (2002) 321-329 

Cell-to-cell and long-distance transport of some plant viruses requires coordinated action of three movement proteins encoded by triple gene block (TGB). The largest of TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and possesses a set of conserved helicase sequence motifs necessary for virus movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and potato virus X, a potexvirus, produced in Escherichia coli had unwinding activity on a partially duplexed RNA, but not DNA substrate. The helicase activity of these proteins was dependent on Mg2+ and ATP. The isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was also able to unwind RNA duplex. 

Kinetics of thermal aggregation of tobacco mosaic virus coat protein 

Kurganov B.I., Rafikova E.R., Dobrov E.N. 

Biochemistry (Moscow) 67 (2002) 525-533 

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 520C in a wide range of protein concentrations, [Plo. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 520C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to Plo (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 420C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]0 and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates (A/Alim; t) (t is time and Alim is the limiting value of A at t --> infinity). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate- limiting stage is the stage of protein molecule unfolding. 

Development of male reproductive organs in an insertion mutant TPD1 of tobacco characterized by extended flowering 

Milyaeva E.L., Gurko N.A., Bavrina T.V., Skurat E.V., Dorokhov Y.L., Romanov G.A. 

Russian Journal of Plant Physiology 49 (2002) 470-477 

A tobacco clone TPD1 (Tobacco Pollen Development), characterized by an extended flowering period, was obtained using a serial agrobacterial transformation of tobacco (Nicotiana tabacum L., cv. Samsun) by constructing a DNA carrying the kanamycin resistance gene inserted into the binary pBin 19 vector. However, the characteristics of the vegetative growth of these plants were similar to those of other tobacco clones and the wild type. In the insertion mutant, pollen was 1.5 times smaller than in the wild type and germinated on the stigmata of neither its own clone nor the wild-type plants. Cytochemical investigation of pollen of the TPD1-mutant did not reveal any activity of respiratory enzymes, succinate dehydrogenase and peroxidase, indicating that the pollen was nonviable. Unlike the wild type, the TPD1 mutant exhibited disturbed trophic interactions within the anther tissues, particularly suppressed starch hydrolysis in the anther wall tissues at the stage of rapidly growing microspores. We conclude that the insertion of T-DNA into the TPD1 gene produced structural and metabolic changes during the development of anther tissues in the mutant clone, resulting in pollen sterility. 

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells 

Zamyatnin A.A., Solovyev A.G., Sablina A.A., Agranovsky A.A., Katul L., Vetten H.J., Schiemann J., Hinkkanen A.E., Lehto K., Morozov S.Y. 

Journal of General Virology 83 (2002) 651-662 

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism. 

 

 

V. STRUCTURE, EXPRESSION AND EVOLUTION OF GENOM

 

 
 
 

Synthesis of Oligonucleotide Derivatives with Aryl(trifluoromethyl)diazirine Moiety for the Photoaffinity Modification of Proteins and Nucleic Acids 

Agapkina Yu.Yu., Agapkin D.V., Zagorodnikov A.V., Alekseev Ya.I., Korshunova G.A., Gottikh M.B.

Russian Journal of Bioorganic Chemistry 28 (2002) 293-299

1-( 4-( 3-( Trifluoromethyl )-3H-diazirin-3-yl )benzamido )-3-O-( 4,4'-dimethoxytrityl )-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)15 were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8-9њC. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA-protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them. 

Molecular evidence of regress in evolution of metazoa

Aleshin V.V., Petrov N.B. 

Zhurnal Obshchei Biologii 63 (2002) 195-208 

Molecular data permit to construct phylogenetic trees independently of morphological characters. It allows to consider their evolution without the frames of a priori hypothesis of regularities of morphological evolution and independently of palaeontological. data. Cladistic analysis of elements of secondary structure of variable areas V7 and V2 in 18S rRNA with different Protozoa as "external" groups shows that Bilateria + Cnidaria are monophyletic, Ctenophora and Porifera are early derivatives of Metazoa, Trichoplax (Placozoa) is a form related to Cnidaria, while Rhombozoa, Orthonectida and Myxozoa were branched within Bilateria. Morphological reduction with losses of any organs and tissues took place many times in early evolution of Metazoa and Bilateria not only in parasitic species. It occurred both at early and late stages of embryonic development and differentiation. Two alternative scenario of morphological degeneration in Trichoplax and the way of their testing are suggested. The similarity of Ctenophora and Calcarea is discussed. Meridional or oblique position of the third cleavage furrow of ovule can be considered as an evidence of their origin from common ancestor. 

Genomics and genosystematics 

Antonov A.S. 

Russian Journal of Genetics 38 (2002) 622-627 

A survey of publications dealing with comparative analysis of genomes shows that modern genomics has naturally evolved from gene systematics- an area whose formation and development was substantially influenced by the scientific school of A.N. Belozersky. 

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues 

Ashapkin V.V., Kutueva L.I., Vanyushin B.F.

FEBS Letters 532 (2002) 367-372

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.

Iodouracil-mediated photocrosslinking of DNA to 

EcoRII restriction endonuclease in catalytic conditions 

Babkina O.V., Chutko C.A., Shashkov A.A., Dzhidzhoev M.S., Eritja R., Gromova E.S. 

Photochemical and Photobiological Sciences (2002) 636-640.

We used a XeCl excimer laser with 50 ns pulses, a frequency of 0.3 Hz and a wavelength of 308 nm in appropriate conditions for the photocrosslinking of EcoRII restriction endonuclease to a 14-mer DNA duplex, containing a 5-iodo-2'-deoxyuridine residue (IdU). IdU replaced the thymidine residue within the EcoRII recognition sequence 5'-CCT/AGG. The binding of EcoRII endonuclease to the IdU-containing DNA duplex was analyzed by gel retardation assay in the presence of Ca2+ or Mg2+ ions. Photocrosslinking of EcoRII to the IdU-containing DNA duplex occurred in a pre-reactive complex formed in the presence of Ca2+ ions. Photocrosslinking yields as a function of time and UV-laser light intensity were studied. 

Disruption of HIV-1 Integrase-DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides

Brodin P., Pinskaya M., Buckle M., Parsch U., Romanova E., Engels J., Gottikh M., Mouscadet J.F.

Biochemistry 41 (2002) 1529-1538 

We recently found that oligonucleotides containing the 6-oxocytosine heterocyclic base are efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al. (2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we demonstrate that the inhibition arises from a noncompetitive mechanism in which the modified oligonucleotide attacks the integrase-DNA complex, leading to its active disruption. This conclusion is based on the following results. First, despite the fact that the respective affinities of a 6-oxocytosine-containing oligonucleotide and of its nonmodified counterpart for integrase were identical, only the modified compound inhibited the enzyme activities. Second, DNA binding and UV cross-linking assays indicated that the 6-oxocytosine-containing oligonucleotide prevented the formation of a stable integrase-DNA complex. Third, the kinetics of the dissociation of the integrase-DNA complex were dramatically accelerated in the presence of the modified ODN, whereas the nonmodified counterpart did not influence the dissociation. This mechanism was supported by the ability of the 6-oxocytosine-containing oligonucleotide to inhibit the strand transfer activity of HIV-1 preintegration complexes in vitro. Disruption of integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides constitutes an original mechanism of integration inhibition, therefore suggesting a strategy for searching for inhibitors of the HIV-1 preintegration complexes.

Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species

Bunik V.I., Sievers C.

European Journal of Biochemistry 269 (2002) 5004-5015

Self-regulation of the 2-oxo acid dehydrogenase complexes during catalysis was studied. Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction. Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD. In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected. In the absence of oxygen, a protein-bound radical concluded to be the thiyl radical of the complex-bound dihydrolipoate was trapped by a-phenyl-N-tert-butylnitrone. Another, carbon-centered, radical was trapped in anaerobic reaction of the complex with 2-oxoglutarate and CoA by 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO). Generation of radical species was accompanied by the enzyme inactivation. A superoxide scavenger, superoxide dismutase, did not protect the enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the inactivation. It was concluded that the thiyl radical of the complex-bound dihydrolipoate induces the inactivation by 1e- oxidation of the 2-oxo acid dehydrogenase catalytic intermediate. A product of this oxidation, the DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the first component of the complex. The inactivation prevents transformation of the 2-oxo acids in the absence of terminal substrate, NAD+. The self-regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes. The data point to a dual pro-oxidant action of the complex-bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD+ + NADH) pool.

N-6-adenine DNA-methyltransferase in wheat seedlings 

Fedoreyeva L.I., Vanyushin B.F. 

FEBS Letters 514 (2002) 305-308 

The N-6-adenine DNA-methyltransferase was isolated from the vacuolar vesicle fraction of wheat coleoptiles. In the presence of S-adenosyl-L-methionine the enzyme de novo methylates the first adenine residue in the TGATCA sequence in the single- or double-stranded DNA substrates but it prefers single-stranded structures. Wheat adenine DNA-methyltransferase (wadmtase) is a Mg2+- or Ca2+-dependent enzyme with a maximum activity at pH 7.5-8.0. Wadmtase seems to be responsible for mitochondrial DNA modification that might be involved in the regulation of replication of mitochondria in plants. 

Point contacts of T7 RNA polymerase in the promoter complex, as determined with phosphate-activated oligonucleotide derivatives 

Filippova S.E., Ivanovskaya M.G., Romanova E.A., Tunitskaya V.L., Kochetkov S.N. 

Molecular Biology 36 (2002) 543-550 

The contacts between phosphate groups of promoter DNA and Lys or His of T7 RNA polymerase (Pot) in the Pot-promoter complex were studied with single- and double-stranded oligonucleotides corresponding to the T7 promoter consensus and containing activated phosphate groups at position +1, +2, or -14 relative to the transcription start. To obtain reactive groups, terminal phosphates were modified with N-hydroxybenzotriazole (HOBT), and internucleotide phosphates were replaced with a trisubstituted pyrophosphate (TSP). The resulting derivatives produced covalent complexes with T7 Pot. Covalent bonding involved His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in the case of TSP at position -14. 

VPg unlinkase, the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: A minimal nucleic moiety of the substrate 

Gulevich A.Y., Yusupova R.A., Drygin Y.F. 

Biochemistry (Moscow) 67 (2002) 615-621 

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5'-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic pan of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg-RNA complex were used: [I-125]Kp-pUpUpGp, [I-125]Kp-pUp, and [I-125]Kp-pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg-RNA). [I-125]K-peptides were unlinked from [ 1251]Kp-pUpUpGp and [I-125)Kp-RNA with similar velocity, but [I-125]Kp-pUp was split much slower. Under the same conditions [125 I]Kp-pU was not dissociated at all. Thus, pUp is a minimal pan of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [I-125]Vpg-pU, [I- 125]VPg-pUp, and [I-125]VPg-pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [I-125]VPg-RNA and [I-125]VPg- pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter- polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA. 

Lymphotoxin-b receptor immune interaction promotes tumor growth by inducing angiogenesis 

Hehlgans T., Stoelcker B., Stopfer P., Muller P., Cernaianu G., Guba M., Steinbauer M., Nedospasov S.A., Pfeffer K., Mannel D.N. 

Cancer Research 62 (2002) 4034-4040 

Growth of solid fibrosarcoma tumors in mice was inhibited by the release of a soluble lymphotoxin-b receptor inhibitor (LTbR-immunoglobulin fusion protein) from the tumor cells. Tumor growth arrest in mice deficient in the ligand LTa(1)b(2) demonstrated the requirement for activation of the LTbR on the tumor cells by host cell-derived LTa(1)b(2). Activation of the LTbR resulted in enhanced release of macrophage inflammatory protein-2. Blocked angiogenesis was revealed in LTbR inhibitor-producing tumor nodules by immunohisto-chemistry and in vivo microscopy. The growth arrest of LTbR inhibitor-producing fibrosarcomas was overcome by forced MIP-2 expression in the tumor cells. Thus, LTbR activation on tumor cells by activated host lymphocytes can initiate a novel proangiogenic pathway leading to organized tumor tissue development. 

Pyrimidine oligodeoxyxylonucleotides form triplexes with purine DNA in neutral medium 

Ivanov S.A., Alekseev Y.I., Gottikh M.B. 

Molecular Biology 35 (2002) 131-139 

Interactions of oligonucleotides comprising ( 1-b-D-2'-deoxy- threo-pentafuranosyl )thymine and (1-b-D-2'-deoxy-threo- pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B. 

A new and efficient method for synthesis of 5 '-conjugates of oligonucleotides through amide-bond formation on solid phase 

Kachalova A.V., Stetsenko D.A., Romanova E.A., Tashlitsky V.N., Gait M.J., Oretskaya T.S.

Helvetica Chimica Acta 85 (2002) 2409-2416 

An efficient method for synthesis of oligonucleotide 5'- conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5'- carboxylic acid function were obtained by use of a novel non- nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2-chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5'-Carboxylate- modified oligonucleotides were efficiently conjugated on solid support under normal peptide-coupling conditions to various amines or to the N-termini of small peptides to yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide conjugates containing an amide linkage between the 5'-end of an oligonucleotide and the N-terminus of a peptide. 

CK2betates gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

Kalmykova A.I., Shevelyov Y.Y., Polesskaya O.O., Dobritsa A.A., Evstafieva A.G., Boldyreff B., Issinger O.G., Gvozdev V.A. 

European Journal of Biochemistry 269 (2002) 1418-1427 

An earlier described CK2betates gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2betates Ig revealed CK2betates protein in Drosophila testes extract. Expression of a CK2betates-b- galactosidase fusion protein driven by the CK2betates promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2betates protein expressed in Escherichia coli revealed properties similar to those of CK2b: (a) CK2betates protein stimulates CK2a catalytic activity toward synthetic peptide: (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2a by polylysine: (c) it is able to form (CK2betates)2 dimers, as well as (CK2a)2(CK2betates)2 tetramers. Using the yeast two- hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes. we demonstrated an association between CK2betates and CK2a. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis. 

X-ray analysis of ribosomes: the static of the dynamic 

Kopylov A.M. 

Biochemistry (Moscow) 67 (2002) 372-382 

This review considers a brief history, comments, and consequences of recent remarkable achievements: X-ray analysis on the level of atomic resolution of structures of bacterial ribosomes, their subunits, and functional complexes. 

DNA Duplexes Containing Photoactive Derivatives of 2'-Deoxyuridine as Photocrosslinking Probes for EcoRII DNA Methyltransferase-Substrate Interaction 

Koudan E.V., Subach O.M., Korshunova G.A., Romanova E.A., Eritja R., Gromova E.S.

Journal of Biomol. Struct. Dyn. 20 (2002) 421-428

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i5dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly268-Met391 region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.

Cyclosporin A blocks the expression of lymphotoxin a, but not lymphotoxin b, in human peripheral blood mononuclear cells 

Kuprash D.V., Boitchenko V.E., Yarovinsky F.O., Rice N.R., Nordheim A., Ruhlmann A., Nedospasov S.A. 

Blood 100 (2002) 1721-1727 

The 2 lymphotoxin subunits LTa (also called tumor necrosis factor b [TNF-b]) and LTb belong to the family of TNF-related cytokines. They form either a soluble homotrimeric ligand (LTa(3)) that binds to and signals through CD120a/b (TNFRp55 and TNFRp75), or a membrane-associated heterotrimeric ligand (LTa(1)b(2)) that binds to and signals through the LTb receptor (LTbR). In mice, LTbR signaling is critical for the maintenance of peripheral lymphoid tissues and optimal immune responses, and its down-regulation results in immunodeficiency. To determine the possible relationship between LT-mediated immunodeficiency and the immunosuppressive effects of cyclosporin A (CsA), we tested the effects of CsA on the expression of LTa and LTb in human peripheral blood mononuclear cells (PBMCs). When PBMCs were stimulated with phorbol myristate acetate/ionomycin or with anti- CD3/anti- CD28, the accumulation of LTa both at mRNA and protein levels was markedly inhibited by CsA. This inhibition is likely due to CsA's effect on the nuclear factor of activated T cell (NFAT) proteins binding to a novel NFAT-binding element at position -490 relative to LTa transcription start. LTb showed a distinct expression pattern and was insensitive to CsA. Thus, in addition to its effects on the expression of other TNF family members, such as TNFa, CD40-L, and CD95-L, CsA can block expression of surface LT complex by selectively inhibiting the expression of the LTa subunit. We propose that LT dysfunction and its downstream effects may contribute to immunosuppressive effects of CsA. 

Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method 

Kubareva E.A., Walter J., Karyagina A.S., Vorob'eva O.V., Lau P.C., Trautner T.

Biotechniques 33 (2002) 526-531

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.

The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans

Kuvardina O.N., Leander B.S., Aleshin V.V., Myl'nikov A.P., Keeling P.J., Simdyanov T.G.

Journal of Eukaryot. Microbiol. 49 (2002) 498-504 

In an attempt to reconstruct early alveolate evolution, we have examined the phylogenetic position of colpodellids by analyzing small subunit rDNA sequences from Colpodella pontica Myl'nikov 2000 and Colpodella sp. (American Type Culture Collection 50594). All phylogenetic analyses grouped the colpodellid sequences together with strong support and placed them strongly within the Alveolata. Most analyses showed colpodellids as the sister group to an apicomplexan clade, albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera, clustered together and consistently branched as the sister group to dinoflagellates as shown previously. These data demonstrate that colpodellids and perkinsids are plesiomorphically similar in morphology and help provide a phylogenetic framework for inferring the combination of character states present in the last common ancestor of dinoflagellates and apicomplexans. We can infer that this ancestor was probably a myzocytotic predator with two heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar ring, and a coiled open-sided conoid. This ancestor also very likely contained a plastid, but it is presently not certain whether it was photosynthetic, and it is not clear whether extant perkinsids or colpodellids have retained the organelle.

Mitochondrial 12S rDNA sequence relationships suggest that the enigmatic bovid "Linh Duong" Pseudonovibos spiralis is closely related to buffalo 

Kuznetsov G.V., Kulikov E.E., Petrov N.B., Ivanova N.V., Lomov A.A., Kholodova M.V., Poltaraus A.B. 

Molecular Phylogenetics and Evolution 23 (2002) 91-94 

The phylogenetic position and taxonomic status of the recently described Southeast Asian endemic bovid Pseudonovibos spiralis were deduced from nearly complete 12S mitochondrial rDNA sequences of this species and Bubalus bubalis alongside 26 sequences of Bovidae from GenBank using Cervus elaphus (Cervidae) as outgroup. Maximum-likelihood analyses performed by PUZZLE and fastDNAml nested P. spiralis at the base of the subtribe buffalo Bovini, suggesting the close relationship of this enigmatic species with buffalo and enabling its distinction into the separate subtribe Pseudonovibovina

Phylogenetic analysis of sequences of the 12S and 16S rRNA mitochondrial genes in the family Bovidae: New evidence 

Kuznetsova M.V., Kholodova M.V., Luschekina A.A. 

Russian Journal of Genetics 38 (2002) 942-950 

The phylogeny of the family Bovidae has been inferred from our data on the 12S and 16S rRNA mtDNA gene sequences and from the results of other authors. A considerable (2460 bp) length of the analyzed fragments of these conserved genes and the use of different methods of cladogram construction allowed us to verify the systematic position of the genera Saiga, Pantholops, Procapra, and Oreamnos. Saigas were shown to be phylogenetically far closer to gazelles than black-tailed gazelles and pygmy antelopes. In general, the genetic analysis data are in agreement with the results of morphological studies. 

Structural Aspects of Interaction of Homeodomains with DNA 

Ledneva R.K., Alexeevskii A.V., Vasil'ev S.A., Spirin S.A., Karyagina A.S.

Molecular Biology (Moscow) 35 (2001) 647-659

This review is devoted to the structural aspects of interaction of homeodomains with DNA. Presented are the list of all homeodomains with known spatial structure and the alignment of their amino acid sequences. The structure of homeodomains and contacts of their amino acid residues with DNA bases and sugar-phosphate backbone are described. The role of water molecules in DNA binding is discussed. Structures of multicomponent protein complexes on DNA including homeodomains are characterized.

Determination of Low Activity of Tritium-Labeled Amino Acids Using Simultaneously Flow Scintillation and Amino Acid Analyzers

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

Radiochemistry 44 (2002) 81-85 

The possibility of measuring a low activity of tritium-labeled amino acids in the eluate from Amino Acid Analyzer 835 (Hitachi, Japan) using a Radiomatic 150TR Flow Scintillation Analyzer (Packard Instrument Co., USA) was studied. Due to stepped variations of pH, ionic strength, and salt concentration in eluting solutions during amino acid separation and utilization of ninhydrin reagent in spectrophotometric measurements of amino acids, special selection of scintillation liquids was necessary. Six scintillation cocktails were tested: ZhS-8 (Reakhim, Ukraine), OptiPhase 'HiSafe' 3 (Wallac Oy, EG&G Co., Finland), Hionic-Fluor, Ultima-Flo AP, Ultima-Flo M, and Ultima Gold (Packard Instrument Co., USA). It was found that Hionic-Fluor and Ultima-Flo AP cocktails are the most appropriate for flow measurements of tritium activity. Under optimal conditions the detection limit with Hionic-Fluor and Ultima-Flo AP was 150 and 100 decays min-1 in the peak of amino acid, respectively. Such a high sensitivity allows utilization of the above analytical system for measurements of amino acid radioactivity to study the structure of proteins and protein complexes by tritium planigraphy. 

Quality control: Proteins and organelles 

Luzikov V.N. 

Biochemistry (Moscow) 67 (2002) 171-183 

This review summarizes materials on the mechanisms of intracellular degradation of proteins whose topogenesis is disturbed at one stage or another. Chaperone and proteolytic systems involved in this process in the endoplasmic reticulum, mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct subcellular compartments of procaryotic cells are considered. The available data suggest that living cells contain numerous systems keeping under control both folding of newly synthesized and newly imported polypeptide chains and their incorporation into heterooligomeric complexes. The point of view is elaborated that organelle formation is controlled not only at the level of individual protein molecules but also at the supermolecular level when whole organelles incapable of carrying out their integral key functions become targets for partial or total elimination. This type of control is realized through an autophagic mechanism involving lysosomes/vacuoles. 

A novel method of introducing hydrophobic moieties into oligonucleotides for covalent and non-covalent immobilization on electrode surfaces 

Oretskaya T.S., Romanova E.A., Andreev S.Y., Antsypovich S.I., Toth C., Gajdos V., Hianik T. 

Bioelectrochemistry 56 (2002) 47-51 

An effective method for the introduction of oleylamine-modified cytidine units into predetermined position(s) of the oligodeoxyribonucleotide (ON) chain during automated ON synthesis has been developed. The high yields of the condensation products upon the introduction of the modified units allow the methods suggested to be used for the synthesis of ONs with two hydrophobic substituents. We also suggest a simple method for obtaining ONs with 5'-terminal hydrophobic linker with free thiol group. The functionality of synthesized ON modified by thiol group and that with hydrophobic spacer for the detection DNA hybridization has been approved in conductometric experiments. 

Amyloid fibrils from the mammalian protein prothymosin a

Pavlov N.A., Cherny D.I., Heim G., Jovin T.M., Subramaniam V. 

FEBS Letters 517 (2002) 37-40 

Mammalian prothymosin a, a small (12 kDa) and extremely acidic protein (pI 3.5), is a member of the growing family of `natively' unfolded proteins. We demonstrate that at low pH ( similar to 3) and high concentrations, prothymosin a is capable of forming regular elongated fibrils with flat ribbon structure 4-5 nm in height and 12-13 nm in width as judged from scanning force and electron microscopy. These aggregates induced a characteristic spectral shift of thioflavin T fluorescence and their circular dichroism spectra were indicative of significant b-sheet content, suggesting formation of classical amyloid. Our findings indicate that natively unfolded proteins may have a general propensity to form amyloid fibrils under conditions inducing partially folded conformations. 

Identification, cloning and characterization of a new DNA- binding protein from the hyperthermophilic methanogen Methanopyrus kandleri 

Pavlov N.A., Cherny D.I., Nazimov I.V., Slesarev A.I., Subramaniam V. 

Nucleic Acids Research 30 (2002) 685-694 

Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially a-helical protein possibly containing a short N-terminal b-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of similar to 43.5+3.5 nm (136+11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins. 

Conditionally neutral phylogenetic markers of major taxa: A new aspect of the evolution of macromolecules 

Petrov N.B., Aleshin V.V. 

Russian Journal of Genetics 38 (2002) 877-894 

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda

Evolutionary relationship between different subgroups of restriction endonucleases 

Pingoud V., Kubareva E., Stengel G., Friedhoff P., Bujnicki J.M., Urbanke C., Sudina A., Pingoud A. 

Journal of Biological Chemistry 277 (2002) 14306-14314 

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type HE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype. 

Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40 S ribosomal subunits to the initiation codon of b-globin mRNA 

Pisarev A.V., Skabkin M.A., Thomas A.A., Merrick W.C., Ovchinnikov L.P., Shatsky I.N. 

Journal of Biological Chemistry 277 (2002) 15445-15451 

AB p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with b-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'- untranslated region linked to the P-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs. 

Ionol (BHT) Produces Superoxide Anion

Smirnova E.G., Lyubimov Yu.I., Malinina T.G., Lyubimova E.Yu., Alexandrushkina N.I., Vanyushin B.F., Kolesova G.M., Yaguzhinsky L.S.

Biochemistry (Moscow) 67 (2002) 1271-1275

In aqueous medium etiolated wheat seedlings release superoxide anion (О2-) Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by О2 -formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce О2 production by the etiolated wheat seedling system.

DNA aptamers as radically new recognition elements for biosensors 

Spiridonova V.A., Kopylov A.M. 

Biochemistry (Moscow) 67 (2002) 706-709 

A fiber-optic biosensor based on DNA aptamers used as receptors was developed for the measurement of thrombin concentration. Anti-thrombin DNA aptamers were immobilized on silica microspheres, placed inside microwells on the distal tip on an imaging optical fiber, coupled to a modified epifluorescence microscope through its proximal tip. Thrombin concentration is determined by a competitive binding assay using a fluorescein- labeled competitor. The biosensor is selective and can be reused without any sensitivity change. The thrombin limit of detection is 1 nM, Sample Volume is 10 ?l, and assay time per sample is 15 min including the regeneration step. 

A comparative thermodynamic study for both natural and artificial RNA/DNA-protein binary complexes 

Spiridonova V., Rassokhin T., Golovin A., Petrova E., Rohzdestvensky T., Pakhomova Y., Kopylov A. 

Bioelectrochemistry 56 (2002) 95-97 

In recent years, Systematic Evolution of Ligands by EXponential enrichment (SELEX) technique has been developed into a fast growing field. In contrast to traditional recognition elements, like antibody, our interests focus on novel molecular recognition elements based on nucleic acids, which are of value both for the therapy and biosensors. A comparative study of thermodynamic for both natural and artificial RNA/DNA-protein complexes would establish bases for a specificity of complex formation. In particular, we have shown that aptamers could be used for a direct measuring of thrombin enzymatic activity in a solution. 

Sensing prothymosin a origin, mutations and conformation with monoclonal antibodies 

Sukhacheva E.A., Evstafieva A.G., Fateeva T.V., Shakulov V.R., Efimova N.A., Karapetian R.N., Rubtsov Y.P., Vartapetian A.B. 

Journal of Immunological Methods 266 (2002) 185-196 

To overcome poor immunogenicity of prothymosin a, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin a antibodies in mice immunized with free human prothymosin a, with prothymosin a coupled to different carriers and with prothymosin a fused to green fluorescent protein was assessed. Fusing prothymosin a to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin a antibodies. From these studies, two highly specific anti-prothymosin a monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin a molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin a that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin a fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti- prothymosin a antibodies obtained were suitable for precipitation of human prothymosin a from HeLa cell lysates and for immunolocalization of the endogenous prothymosin a within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins. 

Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues 

Tumanov A.V., Kuprash D.V., Lagarkova M.A., Grivennikov S.I., Abe K., Shakhov A.N., Drutskaya L.N., Stewart C.L., Chervonsky A.V., Nedospasov S.A.

Immunity 17 (2002) 239-250.

In order to definitively ascertain the functional contribution of lymphotoxin (LT) expressed by B cells, we produced mice with the LTb gene deleted from B cells (B-LTb KO mice). In contrast to systemic LTb deletion, in B-LTb KO mice only splenic microarchitecture was affected, while lymph nodes and Peyer's patches (PP) were normal, except for PP's reduced size. Even though B-LTb KO spleens retained a small number of follicular dendritic cells (FDC) which appeared to be dependent on LPb produced by T cells, IgG responses to sheep red blood cells were markedly reduced. Thus, the organogenic function of B-LTb is almost entirely restricted to spleen, where it supports the correct lymphoid architecture that is critical for an effective humoral immune response. 

Relationships among genera in Saniculoideae and selected Apioideae (Umbelliferae) inferred from nrITS sequences 

Valiejo-Roman C.M., Terentieva E.I., Samigullin T.H., Pimenov M.C. 

Taxon 51 (2002) 91-101 

The internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were newly sequenced for eight species of Umbelliferae (six species from subfamily Saniculoideae: Actinolema macrolema, Astrantia minor, Eryngium giganteum, E. coeruleum, Hacquetia epipactis, and Lagoecia cuminoides, two species from subfamily Hydrocotyloideae: Dickinsia hydrocotyloides and Azorella trifurcata), as well as Hohenackeria exscapa, a species of uncertain position in the family. Phylogenetic analyses of new data, plus previously reported sequences of 52 other species using neighbor-joining, maximum parsimony, and maximum likelihood methods yielded similar results: (1) Actinolema is sister to Astrantia corresponding to Drude's treatments; (2) in Astrantia, molecular divergence is revealed between sects. Astrantiella (A. minor) and Astrantia (A. major, A. maxima); (3) Eryngium appears to be paraphyletic; (4) Hacquetia might be treated as a part of Sanicula; and (5) Lagoecia is very distant from all other Saniculoideae and close to some genera of Apioideae. Our results correspond to matK data previously published: (1) Hohenackeria forms a clade with Bupleurum, in a position near the base of the Apioideae tree; (2) Azorella is sister to a large cluster uniting all Saniculoideae and Apioideae, being slightly closer to them than to the Hydrocotyle-Araliaceae clade; (3) Dickinsia is very distant from phenetically similar Hydrocotyle, falling within a large cluster of Apioideae, but also including Lagoecia and Naufraga

The effects of phytohormones and 5-azacytidine on apoptosis in etiolated wheat seedlings 

Vanyushin B.F., Shorning B.Y., Seredina A.V., Aleksandrushkina N.I. 

Russian Jorrnal of Plant Physiology 49 (2002) 501-506 

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A3, fusicoccin C, and 2,4-D at the concentration of 10-5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10-5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10-2-10-3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 ?g/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes. 

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 (Mjscow) 67 (2002) 67 212-221 

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 at. (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. 

Apoptosis in Etiolated Wheat Seedlings: 1. Ultrastructure of the Apoptotic Cell 

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

Russian Journal of Plant Physiology 49 (2002) 736-745

We studied the process of apoptosis in etiolated wheat (Triticum aestivum L.) seedlings. As a result, an integral pattern of the apoptotic plant cell ultrastructure was established. In the apoptotic cells of the coleoptile, we observed chromatin condensation and margination, an increased density and specific cytoplasm fragmentation accompanied by the appearance of unusual cytoplasmic vesicles containing subcellular organelles, mitochondria in particular, in the vacuoles.

Synthesis of 2 '-O-alkylnucleosides 

Zatsepin T.S., Romanova E.A., Oretskaya T.S. 

Uspekhi Khimii 71 (2002) 586-608 

Methods for the synthesis of 2'-O-alkylnucleosides are reviewed. The known methods are described systematically, their advantages and drawbacks are discussed. 

Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2 '-aldehydes through thiazolidine, oxime, and hydrazine linkages 

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

Bioconjugate Chemistry 13 (2002) 822-830 

2'-Deoxyoligonucleotides and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy, or hydrazide group to give peptide-oligonucleotide conjugates incorporating single or multiple peptides in good yield. The facile conjugation method allows specific coupling in aqueous solution of unprotected oligonucleotides containing aldehyde groups to unprotected N-terminally modified peptides and other small molecules. A 12-mer 2'-O- methyloligoribonucleotide complementary to the HIV-1 TAR RNA stem-loop and containing two conjugated copies of an 8-mer model laminin peptide was hardly affected in TAR RNA binding and showed a similar level of inhibition of HIV-1 Tat-dependent in vitro transcription compared to the unconjugated 2'-O- methyloligoribonucleotide. Advantages of this conjugation method include (1) the ability to attach more than one peptide or other small molecule to oligonucleotide at defined nucleoside residue locations; (2) a conjugation route that does not affect significantly oligonucleotide binding to RNA structures; and (3) three alternative, facile, and mild conjugation reaction types that do not require use of a large excess of peptide reagent. 

Interaction of influenza A virus M1 matrix protein with caspases

Zhirnov O.P., Ksenofontov A.L., Kuzmina S.G., Klenk H.D.

Biochemistry (Moscow) 67 (2002) 534-539 

In this investigation, an ability of influenza A virus M1 matrix protein to bind intracellular caspases, the key enzymes of cell apoptosis, has been examined. Protein-protein binding on polystyrene plates and polyvinyl pyrrolidone membrane was employed for this purpose. Under a comparative study of caspases-3, -6, -7, -8 influenza virus M1 protein specifically bound caspase-8 and weakly bound caspase-7. Using a computer analysis of the N-terminal region of M1 protein, a site similar to the anti-caspase site of baculovirus p35 protein, which inhibits caspases and displays antiapoptotic activity, was identified. These results are in good agreement with the supposition that influenza virus M1 protein is involved in a caspase-8-mediated apoptosis pathway in influenza virus infected cells.

Modern approaches to the synthesis of peptide-oligonucleotide conjugates 

Zubin E.M., Romanova E.A., Oretskaya T.S. 

Uspekhi Khimii 71 (2002) 273-302 

Published data on the methods of chemical synthesis of peptide- oligonucleotide conjugates in solution and on the solid phase are discussed. The known methods are systematised, their advantages and drawbacks are noted. The studies devoted to the synthesis of peptide-oligonucleotide conjugates in solution are arranged according to the type of chemical band between the fragments, while in the case of solid-phase synthesis, according to the method of conjugate preparation (either successive growth of the oligonucleotide and peptide chains on a single polymer support or solid-phase condensation of two presynthesised fragments). 

 

 

VI. ENZYMOLOGY AND BIOTECHNOLOGY

 

 

H+-Pyrophosphatase of Rhodospirillum rubrum - High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation by mersalyl

Belogurov G.A., Turkina M.V., Penttinen A., Huopalahti S., Baykov A.A., Lahti R. 

Journal of Biological Chemistry 277 (2002) 22209-22214 

H+-translocating pyrophosphatase (H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H+-PPase was observed in inner membrane vesicles, where it catalyzed both PPi hydrolysis coupled with H+ transport into the vesicles and PPi synthesis. The hydrolytic activity of H+-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H+-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys185, Cys222, or Cys573 with aliphatic residues had no effect on the activity of H+-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H+-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg2+ and MgPPi protected Cys185 and Cys573 from modification by this agent but not Cys222. Phylogenetic analyses of 23 nonredundant H+-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys222 and includes all known K+-independent H+-PPases, whereas the other incorporates a conserved Cys573 but lacks Cys222 and includes all known K+-dependent H+-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K+ dependence of H+-PPase. 

Enzymatic hydrolysis of b-lactam antibiotics at low pH in a two-phase "aqueous solution water-immiscible organic solvent" system 

Chilov G.G., Svedas V.K. 

Canadian Journal of Chemistry - Revue Canadienne de Chimie 80 (2002) 699-707

The application of the two-phase "aqueous solution - water- immiscible organic solvent" system is suggested not for effective biocatalytic synthesis, but for hydrolytic purposes. Enzymatic hydrolysis of benzylpenicillin and N-phenylacetamidodesacetoxycephalosporanic acid to corresponding antibiotic nuclei 6-aminopenicillanic and 7- aminodesacetoxycephalosporanic acids in a two-phase water-butyl acetate system at pH 3-4 is proposed as an alternative to the biocatalytic hydrolysis in an alkaline medium. An experimental study has been performed and a model has been developed, which describes the influence of pH, phase volume ratio, thermodynamic constants, and initial antibiotic concentration on the effectiveness of their hydrolysis in a two-phase "aqueous solution - water-immiscible organic solvent" system. The thermodynamic evaluation of penicillin G and 7- phenylacetamidodesacetoxycephalosporanic acid hydrolysis at low pH in a two-phase aqueous solution - water-immiscible organic solvent system has demonstrated high practical potential. The suggested approach allows for the exclusion of several technological steps during the transformation of natural b-lactam antibiotics to their semi-synthetic analogues: alkaline extraction of the biosynthetic antibiotic from butylacetate followed by its enzymatic hydrolysis at pH 7.5-8.0 and further acidification of the reaction mixture, which results in the precipitation of the antibiotic nucleus. Experimental observations also revealed a specific feature of this process: the kinetic supersaturation of the antibiotic nucleus slows down the attainment of the equilibrium, which should be taken into account when further developing this approach. 

Single-turnover kinetics of Saccharomyces cerevisiae inorganic pyrophosphatase 

Halonen P., Baykov A.A., Goldman A., Lahti R., Cooperman B.S.

Biochemistry 41 (2002) 12025-12031

Soluble inorganic pyrophosphatase (PPase), which converts inorganic pyrophosphate (PPi) into usable phosphate, is almost universally present as a central enzyme of phosphorus metabolism and uses divalent metal ion as a necessary cofactor. PPase from Saccharomyces cerevisiae (Y-PPase) is the best studied with respect to both structure and mechanism. Here we report the first combined use of stopped flow and quenched flow techniques to study the PPase reaction in both the forward (PPi hydrolysis) and back (PPi synthesis) directions. The results of these studies permit direct comparison of different divalent metal-ion effects (Mg2+, Mn2+, Co2+) on microscopic rate constants at pH 7.0. For the Mn-enzyme, on which all of the high-resolution X-ray studies have been conducted, they demonstrate that the rate-determining step changes as a function of pH, from hydrolysis of enzyme-bound PPi at low pH to release of the more tightly bound Pi at high pH. They also provide evidence for two kinetically important forms of the product complex EM4(Pi)2, supporting an earlier suggestion based on crystallographic evidence, and allow informed speculation as to the identities of acidic and basic groups essential for optimal PPase catalytic activity.

The Effect of Proteolytic Removal of the C-Terminal Fragment of Rhodopsin on Its Ability to Activate Visual Cascade 

Komolov K. E., Senin I. I., Filippov P. P.

Russian Journal of Bioorganic Chemistry 28 (2002) 14-19

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges catalytic activity of the resulting truncated rhodopsin compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. 

The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate 

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

Biochemical and Biophysical Research Communications 294 (2002) 155-160 

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the (NH)-H-1'-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N-1'-H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding. 

Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase 

van Langen L.M., Janssen M.H.A., Oosthoek N.H.P., Pereira S.R.M., Svedas V.K., van Rantwijk F., Sheldon R.A. 

Biotechnology and Bioengineering 79 (2002) 224-228 

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier. 

Three-dimensional domain swapping in homooligomeric proteins and its functional significance

Nagradova N.K.

Biochemistry (Moscow) 67 (2002) 839-849 

In the process of oligomeric structure formation through a mechanism of three-dimensional domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical monomer. The swapped "domain" can represent an entire tertiary globular domain or an element of secondary protein structure, such as an (x-helix or a P-strand. Different examples of three-dimensional domain swapping are reviewed; the functional importance of this phenomenon and its role in the development of new properties by some proteins in the process of evolution are considered. The contribution of three- dimensional domain swapping to the formation of linear protein polymers and amyloids is discussed. 

Regulation of leukotriene synthesis by arachidonic acid in human polymorphonuclear leukocyte adhesive interactions is dependent on the presence of albumin 

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

Cell Biol. Int. 26 (2002) 993-1002

We have previously demonstrated that the pretreatment of polymorphonuclear leukocytes (PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment and inhibits calcium ionophore A23187-stimulated leukotriene (LT) synthesis. Here, we examined the effects of extracellular arachidonic acid (AA) and albumin on attachment and LT synthesis in the interaction of PMNs with both collagen-coated surfaces and human umbilical vein endothelial cell (HUVEC) monolayers. Suramin decreased the release of radiolabelled AA and 5-lipoxygenase metabolites by [14C-AA]-prelabelled PMNs stimulated with A23187, with and without human serum albumin (HSA) in the culture medium. Addition of 1 mM AA together with calcium ionophore stimulated the release of endogenous AA to the same level as control and Suramin-pretreated cells, but attachment was unaffected and LT synthesis was still inhibited with Suramin treatment. Using 24 mM AA, regulation of LT synthesis was dependent on the presence of HSA in the medium. Without HSA, 24 mM AA induced detachment of PMNs and increased LT synthesis in Suramin-treated cells above the control level. In the presence of HSA, 24 mM AA did not influence PMN attachment or abolish Suramin-induced inhibition of LT synthesis. These results suggest that tight attachment of PMNs to a solid surface leads to decreased LT synthesis during subsequent stimulation of the cells by A23187 in the presence or absence of exogenous substrate.

Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D- glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Roitel O., Ivinova O., Muronetz V., Nagradova N., Branlant G. 

Biochemistry 41 (2002) 7556-7564 

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type. 

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site 

Salminen A., Parfenyev A.N., Salli K., Efimova I.S., Magretova N.N., Goldman A., Baykov A.A., Lahti R. 

Journal of Biological Chemistry 277 (2002) 15465-15471 

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 ?M, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH greater than or equal to 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p- hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys83), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg2+ cofactor, P-i product, and the PPi analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas kcat remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys83 residue in the dimer.

Effect of the Structure of Substituent in the Substrate Molecule on Nuclear-Chemical Synthesis of Halonium Compounds

Shchepina N.E., Nefedov V.D., Toropova M.A., Badun G.A., Avrorin V.V., Fedoseev V.M.

Radiochemistry 44 (2002) 378-379

Nuclear-chemical synthesis of halonium compounds was studied, and the influence exerted by the structure of substituent [R = H, 4-Br, 4-CH3, 2-CH3, 2,4,6-(CH3)3, 4-NO2, 2,3,4,5,6-F] in substituted halobenzene substrate molecule on the yield of the onium compound was examined. Electron-withdrawing (bromo, nitro, perfluoro) and sterically shielding (2,4,6-trimethyl) substituents fully suppress formation of the corresponding onium compound. At the same time, the electron-donor methyl group in the p-position of the ring (s = -0.17) activates the lone electron pair of the halogen, which increases the yield of the onium compound. This is especially important in the case of unknown diarylfluoronium derivatives which can be prepared today by a nuclear-chemical procedure only.

Arachidonic acid and docosahexaenoic acid suppress thrombin- evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid liberation. 

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

Journal of Neurochemistry 82 (2002) 1252-1261 

Arachidonic (AA) and docosahexaenoic acid (DHA) are the major polyunsaturated fatty acids (PUFAs) in the brain. However, their influence on intracellular Ca2+ signalling is still widely unknown. In astrocytes, the amplitude of thrombin- induced Ca2+ response was time-dependently diminished by AA and DHA, or by the AA tetraynoic analogue ETYA, but not by eicosapentaenoic acid (EPA). Thrombin-elicited Ca2+ response was reduced (20-30%) by 1-min exposure to AA or DHA. Additionally, 1-min application of AA or DHA together with thrombin in Ca2+-free medium blocked Ca2+ influx, which followed after readdition of extracellular Ca2+. EPA and ETYA, however, were ineffective. Long-term treatment of astrocytes with AA and DHA, but not EPA reduced the amplitude of the thrombin-induced Ca2+ response by up to 80%. AA and DHA caused a comparable decrease in intracellular Ca2+ store content. Only DHA and AA, but not EPA or ETYA, caused liberation of endogenous AA by cytosolic phospholipase A2 (cPLA2). Therefore, we reasoned that the suppression of Ca2+ response to thrombin by AA and DHA could be due to release of endogenous AA. Possible participation of AA metabolites, however, was excluded by the finding that specific inhibitors of the different oxidative metabolic pathways of AA were not able to abrogate the inhibitory AA effect. In addition, thrombin evoked AA release via activation of cPLA2. From our data we propose a novel model of positive/negative-feed-back in which agonist- induced release of AA from membrane phospholipids promotes further AA release and then suppresses agonist-induced Ca2+ responses. 

Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane 

Shishkov A.V., Ksenofontov A.L., Bogacheva E.N., Kordyukova L.V., Badun G.A., Alekseevsky A.V., Tsetlin V.I., Baratova L.A. 

Bioelectrochemistry 56 (2002) 147-149 

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04 + 0.02 A-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15 + 0.05 A-1) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane. 

Isolation and properties of noncovalent complex of transketolase with RNA 

Solovjeva O.N. 

Biochemistry (Moscow) 67 (2002) 667- 

A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified. It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA. Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme. Conditions were chosen for complex dissociation and free enzyme isolation. In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5- fold, respectively The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased. The latter was necessary for the complete manifestation of the enzymatic activity. Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme. 

Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin 

Ubl J.J., Grishina Z.V., Sukhomlin T.K., Welte T., Sedehizade F., Reiser G. 

American Journal of Physiology - Lung Cellular and Molecular Physiology 282 (2002) L1339-L1348 

Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH2 terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca2+ imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca2+ response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U- 73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca2+ level nor PAR-2-elicited Ca2+ response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca2+ response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin. 

Hexameric, Trimeric, Dimeric, and Monomeric Forms of Inorganic Pyrophosphatase from Escherichia coli

Vainonen Yu.P., Kurilova S.A., Avaeva S.M.

Russian Journal of Bioorganic Chemistry 28 (2002) 385-391

The conditions were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia coli inorganic pyrophosphatase from its native hexameric form. Interconversions of the oligomers were studied, and rate constants for their dissociation and association were determined. All forms were found to be catalytically active, with the activity decreasing in the following order: hexamer-trimer-dimer-monomer. The activity of trimeric and dimeric forms was high enough to study and to compare their catalytic properties. The monomeric form of the enzyme was unstable. 

Temperature-induced selective death of the C-domain within angiotensin-converting enzyme molecule 

Voronov S., Zueva N., Orlov V., Arutyunyan A., Kost O. 

FEBS Letters 522 (2002) 77-82 

Somatic angiotensin-converting enzyme (ACE) consists of two homologous domains, each domain bearing a catalytic site. Differential scanning calorimetry of the enzyme revealed two distinct thermal transitions with melting points at 55.3 and 70.50C. which corresponded to denaturation of C- and N- domains, respectively. Different heat stability of the domains underlies the methods of acquiring either single active N- domain or active N-domain with inactive C-domain within parent somatic ACE. Selective denaturation of C-domain supports the hypothesis of independent folding of the two domains within the ACE molecule. Modeling of ACE secondary structure revealed the difference in predicted structures of the two domains, which, in turn, allowed suggestion of the region 29133 in amino acid sequence of the N-part of the molecule as responsible for thermostability of the N-domain. 

Quantitative characterization of the nucleophile reactivity in penicillin acylase-catalyzed acyl transfer reactions 

Youshko M.I., Chilov G.G., Shcherbakova T.A., Svedas V.K. 

Biochimica et Biophysica Acta - Proteins Proteomics 1599 (2002) 134-140 

Nucleophile reactivity of two most known nuclei of penicillins and cephalosporins, 6-aminopenicillanie (6-APA) and 7- aminodesacetoxycephalosporanic (7-ADCA) acids, was quantitatively characterized. In penicillin acylase (PA)- catalyzed acyl transfer reactions the relative reactivity of the added nucleophile compared to the water (i.e. nucleophile reactivity) is defined by two complex kinetic parameters b(0) and g, and depends on the nucleophile concentration. In turn, parameters b(0) and g were shown to be dependent on the structure of both reactants involved: nucleophile and acyl donor. Analysis of the kinetic scheme revealed that nucleophile reactivity is one of a few key parameters controlling efficiency of PA-catalyzed acyl transfer to the added nucleophile in an aqueous medium, Computation of the maximum nucleophile conversion to the product using determined nucleophile reactivity parameters in the synthesis of three different antibiotics, ampicillin, amoxicillin and cephalexin, showed good correlation with the results of corresponding synthetic experiments, Suggested approach can be extended to the quantitative description and optimization of PA-catalyzed acyl transfer reactions in a wide range of experimental conditions. 

Penicillin acylase-catalyzed ampicillin synthesis using a pH gradient: A new approach to optimization 

Youshko M.I., van Langen L.M., de Vroom E., van Rantwijk F., Sheldon R.A., Svedas V.K. 

Biotechnology and Bioengineering 78 (2002) 589-593 

The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-H-phenylglycine amide (D-PGA) to 6- aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (VsNH) is pH-dependent and exhibits a maximum at pH 6.5-7.0 for a saturated solution of 6APA; 2) at a fixed 6-APA concentration below saturation, VsNH increases with decreasing pH. Optimum synthetic efficiency could, therefore, be achieved by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing the pH to 6.3 in the course of 6- APA consumption. A conversion of 96% of 6-APA and 71% of DPGA into ampicillin was accomplished in an opitimized procedure, which significantly exceeds the efficiency of enzymatic synthesis performed at a constant pH of either 7.0 or 6.3. 

Determination of Mn(II) and Co(II) with Arsenazo III 

Zyryanov A.B., Baykov A.A. 

Biochemistry (Moscow) 67 (2002) 635-639 

Complex formation between Arsenazo III and Mn2+ and Co2+ at equilibrium has been investigated at PH 7.2, and the stoichiometry and stability of the complexes have been determined. The data indicate that Arsenazo III is suitable for determination of Mn2+ and Co2+ on the mmolar scale. The dissociation constants of the phosphate complexes of Mn2+ and Co2+ at PH 7.2 were estimated with Arsenazo III as 3.6 and 10 mM, respectively.