M.V. Lomonosov Moscow State University

A.N. Belozersky Institute

of Physico-Chemical Biology

ANNUAL REPORT

1999

 

Content

BIOLOGY OF CELL AND CELL ORGANELLES

BIOENERGETICS AND PHOTOSYNTHESIS

MOLECULAR VIRUSOLOGY

GENOM STRUCTURE, EXPRESSION AND EVOLUTION

ENZIMOLOGY AND BIOTECHNOLOGY
 
 

In HeLa, PK, 3T3, PtKi cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.
 
 

The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK cells during their adaptation to hypotonic medium was studied. The recondensation process was found to be slowed down by modification of plasma membrane with low concentrations of glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The effect of glutaraldehyde can be rapidly reversed by the addition of Ca²⁺-ionophore A23187. Intracellular Ca²⁺ measurements show that the adaptation to hypotonic shock is accompanied by restoration of free Ca concentration, whereas the delay of chromatin condensation in glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the role of low concentration of Ca²⁺ in chromatin compactization in vivo are discussed.
 
 

Studies of the mechanism for proteolysis of 13S globulin, the major reserve protein of buckwheat (Fagopyrum esculentum Moench) seeds, during its mobilization in the process of germination, are discussed. A regulatory system of proteolysis in mature seeds in the course of externally imposed dormancy is also considered. Proteolysis of 13S globulin is affected by the successive action of several proteolytic enzymes belonging to different classes. The first step occurs at the early stages of germination, when metalloproteinase present in seeds modifies the protein via limited proteolysis. Then, the modified protein is subjected to deep digestion by cysteine proteinase and carboxypeptidase, as both enzymes appear in the germinating seeds. The aspartyl proteinase present in dry seeds can be also involved in this process. The endogenous inhibitor of proteinases, the concentration of proteolysis products, intracellular pH, the concentrations of the ions of bivalent metals, and abscisic acid control the progress of 13S globulin proteolysis. In addition to the inhibitors of endogenous proteases, buckwheat seeds contain the inhibitors of exogenous proteolytic enzymes, which belong to two groups, anionic and cationic proteases. These inhibitors are capable of suppressing pathogenic microorganisms, because they also inhibit fungal and bacterial proteases. Thus, these inhibitors evidently participate in the plant defense system.
 
 

The properties of myosin modified at the SH2 group (Cys-697) were studied and compared with the previously reported properties of myosin modified at the SH1 group (Cys-707). 4-[N-[(iodoacetoxy)ethyl]-N methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) was used for selective modification of the SH2 group on myosin. SH2-labeled heavy meromyosin (SH2-HMM), similar to SH1-labeled HMM (SH1-HMM), did not propel actin filaments in the in vitro motility assays. SH1- and SH2-HMM produced similar amounts of load in the mixtures with unmodified HMM; the sliding speed of actin filaments gradually decreased with an increase in the fraction of either one of the modified HMMs in the mixture. In analogy to SH1-labeled myosin subfragment 1 (SH1-S1), SH2-labeled S1 (SH2-S1) activated regulated actin in the in vitro motility assays. SH2 modification inhibited Mg-ATPase of S1 and its activation by actin. The weak binding of S1 to actin was unaffected whereas the strong binding was weakened by SH2 modification. Overall, our results demonstrate similar behavior of SH1 - and SH2-modified myosin heads in the in vitro motility assays despite some differences in their enzymatic properties. The effects of these modifications are ascribed to the location of the SH1-SH2 helix relative to other functional centers of S1.

Mitochondria are critical for the function of nerve terminals, as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca²⁺ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i. e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 nm distance from the edge of the active zone (on average 1. 2 per active zone, range of l-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4. 5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.
 
 

It is known that in oocytes of P. lividus the nucleus contains a single giant nucleolus of unusual structure where intensive rRNA synthesis occurs. However, the questions of structural and functional relationships between the nucleolar compartments and the sites of active rRNA transcription still remain open. In the present work, we studied the chromatin organization in the nucleoli of P. lividus oocytes using Feulgen's and osmium amine staining procedures. Our results indicate that nucleolar chromatin of small (immature) oocytes differs from that in large mature oocytes. At the early stage of development, the DNA filamentous network 0.2-0.5 m m in diameter is formed in the nucleoli. Profound changes in the structure of the nucleolar DNA are observed in the course of oogenesis. Thus, at the late stage of development, the nucleolar chromatin forms characteristic ring-like structures, which indicates a non-uniform distribution of active ribosomal genes. A model of structural organization of the P. lividus nucleolus is discussed.

The marine mollusk Clime unwina when swimming can stabilize different body orientations in the gravitational field. Here we describe one of the modes of operation of the postural network in Clione—maintenance of the vertical, head-up orientation. Experiments were performed on the CNS-statocyst preparation. Spike discharges in the axons of different types of neurons were recorded extracellularly when the preparation was rotated in space through 360° in different planes. We characterized the spatial zones of activity of the tail and wing motor neurons as well as of the CPB3 interneurons mediating the effects of statocyst receptor cells on the tail motor neurons. It was found that the activity of the tail motor neurons increased with deviation of the preparation from the normal, rostral-side-up orientation. Their zones of activity were very wide (—180°). According to the zone position, three distinct groups of tail motor neuron (T1-T3) could be distinguished. The Tl group had a center of the zone near the ventral-side-up orientation, whereas the zones of T2 and T3 had their centers near the left-side-up and the right-side-up positions, respectively. By comparing the zone of activity with the direction of tail bending elicited by each of the groups, one can conclude that gravitational reflexes mediated by the Tl, T2, and T3 groups will evoke turning of the animal toward the head-up orientation. Two identified wing motor neurons, 1A and 2A, causing the wing beating, were involved in gravitational reactions. They were activated with the downward inclination of the ipsilateral side. Opposite reactions were observed in the motor neurons responsible for the wing retraction. A presumed motor effect of these reactions is an increase of oscillations in the wing that is directed downward and turning of Clione toward the head-up orientation. Among the CPB3 interneurons at least four groups could be distinguished. In three of them (1N1. 1N2, and IN3), the zones of activity were similar to those of the three groups (T 1, T2, and T3) of the tail motor neurons. The group IN4 had the center of its zone in the dorsal-side-up position; a corresponding group was not found among the tail motor neurons. In lesion experiments, it w as found that gravitational input mediated by a single CPB3 interneuron produced activation of its target tail motor neurons in their normal zones, but the strength of response was reduced considerably. This finding suggests that several interneurons with similar spatial zones converge on individual tail motor neurons. In conclusion, because of a novel method, activity of the neuronal network responsible for the postural control in Clione was characterized in the terms of gravitational responses in different neuron groups comprising the network.
 
 

The effect of the medium composition on the quantitative and qualitative composition of extracellular proteases of the fungus Alternaria alternata grown in a protein-containing medium was studied. The addition of inorganic nitrogen to the growth medium substantially reduced extracellular proteolytic activity. The range of extracellular proteases was found to depend on the kind of sugar added to the medium. A particular sugar could not only affect the content of extracellular enzymes but also induce the synthesis of new proteases. The range of extracellular proteases produced by micromycetes grown in the protein-containing medium was species-specific. Of the fungi used in this study, Ulocladium artrum exhibited the widest spectrum of extracellular proteases, whereas the spectrum of proteases secreted by Aspergillus penicillosa was the narrowest. Presumably, the high variability of the quantitative and qualitative composition of the extracellular proteases of mycelial fungi is one of the mechanisms providing for their rapid adaptation to varying environmental conditions.
 
 

The cDNA encoding the protein kinase pEg2 was originally cloned through a differential screening performed during the early development of Xenopus laevis. pEg2 orthologues were found in various organisms and were classified in a new family of oncogenic mitotic protein kinases named 'aurora/Ipll-related kinases' after the Drosophila melanogaster gene aurora and the Saccharomyces cerevisw gene Ipl1. The catalytic activity of pEg2 is necessary for the mitotic microtubule spindle formation in Xenopus laevis egg extracts. The addition of a dominant negative form of pEg2 to in vitro spindle assembly assays leads to monopolar spindles generated by a defect of centrosome separation. In Xenopus cultured cells, pEg2 was confined around the pericentriolar material once centrosomes were duplicated. The centrosome localization does not depend on the presence of microtubules. However, in vitro, the protein binds to taxol-stabilized microtubules independently of its kinase activity. During mitosis the location of the protein changes, in metaphase the kinase localizes on the microtubules at the poles of the mitotic spindle whereas it is not present on astral microtubules. This localization persists until the segregation of the chromosomes is completed. The presence of the kinase on the spindle may reveal another yet unknown function.
 
 

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k_ass) was 2.2 10⁶ M^-1 sec^-1 and the dissociation rate constant (k_off) of the enzyme--inhibitor complex was 3.5 10^-3 sec^-1; the inhibition constant K_i was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.
 
 

Microtubules (MTs) are essential for the maintenance of asymmetric cell shape and motility of fibroblasts. MTs are considered to function as rails for organelle transport to the leading edge. We investigated the relationship between the motility of Vero fibroblasts and saltatorymovements of particles in their lamella. Fibroblasts extended their leading edges into the experimental wound at a rate of 20±11 m m/h. Intracellular particles in the front parts of the polarized fibroblasts moved saltatorily mainly along the long axis of the cells. MT depolymerization induced by the nocodazole at a high concentration (1.7 m M) resulted in the inhibition of both fibroblast motility and saltatory movements of the particles. Taxol (1 m M) inhibited the fibroblast locomotion but not the saltatory movements. The saltatory movement pattern was disorganized by taxol by decreasing the portion of longitudinal saltations and consequently by increasing the part of saltations perpendicular to the cell long axis. This effect may be explained by disorganization of the MT network resulting from the inhibition of dynamic instability. To further investigate the relationships between the MT dynamics instability, saltatory movements, and fibroblast locomotion, we treated fibroblasts with microtubule drugs at low concentration (nocodazole, 170 nM; vinblastine, 50 nM; and taxol, 50 nM). All these drugs induced rapid disorganization of the saltatory movements and decreased the rate of cell locomotion. Simultaneously, the amount of acetylated (stable) MTs increased. The treatment also induced reversible changes in the actin meshwork. We suggest that decrease in the fibroblast locomotion rate in the case of MT stabilization occurred because of the appearance of numerous free MTs. Saltations along free MTs are poorly organized and, as a result, the number of organelles reaching the fibroblast leading edge decreases.
 
 

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.
 
 

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning colorimetry. The excess heat capacity vs temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem. 70 (1997) 125]. The energy activation value has been calculated to be 461.0± 0.7 kJ/mol.
 
 

Ethylene oxide and propylene oxide block copolymers (pluronics) are widely known as agents that promote drug penetration across biological barriers. We have studied the interaction of normal and malignant blood cells with pluronics L61 and P85 that have different hydrophobicity. SP2/0 myeloma cells accumulated pluronics while normal cells adsorb most of the polymer on the surface. Interaction of pluronics with cells resulted in drastic changes of membrane microviscosity. Tumor cell membrane microviscosity decreased after pluronics adsorption, in contrast to normal cells, whose membrane microviscosity was enhanced. We suppose that sensitivity of tumor cell membrane microviscosity to the pluronics action correlates with its permeability for molecular substances.

Using specific autoimmune sera to the nucleolar protein fibrillarin and monoclonal antibodies to B23/nucleophosmin, we localized early and late nucleolar rRNA-processing factors in cycling human HeLa and pig PK cells. It was shown that, at the electron microscopic level, fibrillarin was located over the nucleolar fibrillar compartment, but was absent in the fibrillar centres. During mitosis, fibrillarin was located within the same domains as B23, namely, the cytoplasm, the perichromo-somal layer, prenucleolar bodies, and the nucleolar cytoplasmic derivatives, but the kinetics of the two proteins during mitosis was essentially different. Thus, fibrillarin dissociated from the nucleolar remnant at prophase of mitosis or following actino-mycin D treatments after B23, but was found to be more prominent within the peri-chromosomal layer at metaphase, and earlier migrated to the reassembled nucleoli at telophase. In contrast to B23, fibrillarin was found to be resistant to the treatment with 2 M NaCl.
 
 

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of loach fish Misgurnus fossilis or from rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton Centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization is speculated.

Neural network for rhythmic wing movements in the swimming mollusc Clione limacinu is a well-studied system. After nerve transaction the efferent wing neurons cannot reach muscles and consequently display intensive central sprouting. In the present work it was shown that two types of efferent neurons with different neurotransmitters: acethylcholinergic locomotor motoneurons and serotonergic modulatory efferent neurons - when deprived of their normal targets, release their neurotransmitter intended for peripheral muscles, in the unusual compartment—neuropile. Such 'unauthorized' release of neurotransmitter may cause nervous system dysfunctions in the damaged brain of other animals.
 
 

Gene 18 of bacteriophage T4 encodes the contractile protein of the tail sheath. Previous work has shown that the full-length recombinant gene product (gp) 18 of 658 amino acid residues assembles in Escherichia coli cells into a long polysheath structure. However, the gpl8 mutants truncated at the N-termini form insoluble aggregates similar to inclusion bodies. In this study, six plasmid vectors expressing the recombinant gpl8 proteins truncated at the C-termini have been constructed. The CD 58, CD 129, CD 152, C[gl]72, CD 248, and CD 287 proteins contain 600, 529, 506, 486, 410, and 371 residues of the full-length gpl8 molecule, respectively. All the recombinant proteins were soluble and, except for the CD 287 mutant, were assembled into polysheath-related structures. Electron microscopy of negatively stained purified proteins was performed and the resulting images were analyzed by computing their Fourier transforms. The CD 58 and CD 129 mutants, in addition to forming common contracted-type polysheath structures, assembled into thinner filaments that we called "noncontracted polysheaths" (NCP). The CD 152, CD 172, and CD 248 proteins assembled into the NCP type only. Image processing showed that the NCP filaments significantly differ from both extended sheaths of T4 particle and polysheaths. The structure of the NCP filaments might correspond to the transitional helices postulated by Moody (J. MoL Biol, 1973, 80, 613-636) that appeared during the process of tail contraction. Our results suggest that a short region at the C-terminus of the CD 129 protein determine the contractile properties of the gpl8 molecule. The shortest, the CD 287 protein, does not assemble into regular structures, thus indicating that a sequence's stretch in the C-end of the CD 248 mutant might be responsible for polymerization of gpl8.
 
 

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the P_i analogs beryllium fluoride (BeF_x) and aluminum fluoride (AlF₄^-) are stable analogs of the myosin ATPase intermediates M*ATP and M**ADP Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 ADP BeF_x and S1 ADP AlF₄^- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 ADP BeF_x occurs much faster (3-4 min) than that of S1 ADP AlF₄^- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeF_x (during 1 min) than after addition of AlF₄^- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 ADP BeF_x and S1 ADP AlF₄^- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 ADP BeF_x or S1 ADP AlF₄^- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a “weak” binding of the S1 ADP BeF_x and S1 ADP AlF₄^- complexes to F-actin occurs without the subsequent transition of the “weak” binding state to the “strong” binding state. Comparison of the light scattering kinetic curves shows that S1 ADP AlF₄^- binds to F-actin faster than S1 ADP BeF_x does: the second-order rate constants for the “weak” binding to F-actin are (62.8 ± 1.8) 10⁶ M^-1sec^-1 in the case of S1 ADP AlF₄^- and (22.6 ± 0.4) 10⁶ M^-1 sec^-1 in the case of S1 ADP BeF_x. We conclude that the stable ternary complexes S1 ADP BeF_x and S1 ADP AlF₄^- can be successfully used for kinetic studies of the “weak” binding of the myosin heads to F-actin.
 
 

Behavior of epitheliocytes and fibroblasts on special discontinuous substrata (metallic grids with square openings of 45x45 m m²) was examined in order to compare the ability of these cells to spread in two mutually perpendicular directions and to stretch over the void spaces. Two cell types with typical fibroblastic morphology, the AGO 1523 line of human foreskin fibroblasts and secondary cultures of mouse embryo fibroblasts, and three cell types with typical epithelial morphology, primary mouse hepatocytes, the IAR-2 line of rat liver cells and the MDCK line of canine kidney epithelial cells (clone 20) were used. We also examined the epitheliocytes (MDCK cells, clone 20) transformed to fibroblast-like morphology by treatment with hepatocyte growth factor/scatter factor (HGF/SF). Time-lapse video microscopy, scanning electron microscopy and immunofluorescence microscopy were used to examine cell reorganizations at various stages of spreading. It was found that early stage of spreading of fibroblasts and epitheliocytes were similar: the cell spread along two bars, perpendicular to each other (bar and crossbar), with the formation of a small triangular lamellar cytoplasm stretched over the opening. Later central parts of the bodies of the fibroblasts retracted from the bars so that the cells remained attached only by their polar lamellae. Successive expansions and partial retractions of these lamellae led to elongation of the cell body crossing several openings of the grid. Epitheliocytes, in contrast to fibroblasts, at the late stages of spreading did not retract their bodies and did not contract polar lamellae. As a result, their central lamellae stretched progressively over the openings. As a result of the treatment of MDCK epitheliocytes with HGF/SF the behavior of the cells on the grids became similar to that of fibroblasts. It is suggested that these distinct spreading patterns of epitheliocytes and fibroblasts are due to the type-specific differences in the actin-myosin cortex. Experiments with microtubule-specific drugs, colcemid and taxol, indicate that the organization of this cortex is under microtubular control.
 
 

The dynamics of chondriome in the ovogenesis of the sea urchin Paracentrotus lividus was studied. Growing oocytes 20-30, 50-60 and 90-100 m m in diameter ("small", "medium-sized" and "large", respectively) and mature eggs were used for the ultrastructural and stereological analysis of mitochondria. Linear parameters of mitochondria (length and thickness) were measured on 3-D reconstruction of serial ultrathin sections using the software developed in the laboratory. The following transformations of chondriome structure were shown to occur during ovogenesis: (1) the number of mitochondria (MT) increases with the growth of cytoplasmic compartment; (2) the modal length of MT increases from 0.5 m m in small oocytes to 1 m m in large ones and decreases again to 0.5 m m in the egg; this process is accompanied by changes in the relative number of spherical MT which decreases in medium-sized oocytes and subsequently rises again in the egg; (3) in medium-sized oocytes, dumb-bell-shaped MT appear first, the number of these MT reaching the maximum to the stage of large oocytes. In mature eggs, the dumb-bell-shaped MT are absent; (4) in small and medium-sized oocytes, the orthodox conformation of MT is observed, in contrast to MT with a condensed matrix in large oocytes and eggs; (5) in mature eggs, mitochondrial clusters containing 10 to 20 MT of various size are formed. Based on the data obtained, we suggested that during ovogenesis of the sea urchin, specific differentiation of the chondriome is induced which leads to the increase in the quantity of MT via multiple division acts, while restricting the MT growth and variability of their shape.
 
 

In the interphase nucleus chromosomes are tightly associated with the nuclear envelope (NE) through special granular chromatin particles termed anchorosomes. It remains unclear whether anchorosomes represent constant nuclear structures, persisting throughout the cell cycle, or they appear only in the interphase during the formation of contacts between the chromosomes and NE. In other words, whether specific NE interaction sites do exist in chromosomes or any region can form anchorosome.

In this work, we used micrononucleated PK cells, in which almost every micro-nucleus (MN) is formed by a single chromosome. The spatial distribution and quantitative characteristics of the anchorosomal layer in MN was studied using stereological analysis and three-dimensional computer reconstruction. It was shown that in cells with about 30 MN, the total surface area of NE reaches about 355 m m2, whereas in normal mononuclear cells it is 110 m m2. Hence, the NE surface increases 3-fold during MN formation. In contrast to normal cells, only 80% of the NE surface in MN is covered with anchorosomes, i.e., the total surface area of the anchorosomal layer increases by a factor of 2.5. The 3D reconstruction has demonstrated highly random distribution of anchorosome-free zones, the distribution patterns varying in individual MN.

These findings are thought to be evidence for the existence of a limited number of specific chromosomal sites potentially capable of forming contacts with NE.
 
 

B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis. B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase both proteins are colocallzed at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilization.
 
 

In the pteropod mollusc Clione limacina, two different groups of motoneurons innervate two physiologically identical wing muscles (dorsal and ventral). When motoneuron axons are crushed in the nerve of whole animals’ regeneration starts. In its course motoneurons initially project to both the correct and incorrect muscles. Then incorrect connections and neurites are eliminated and the original innervation is restored. Here we investigated neuromuscular regeneration when one of the muscles was removed or the muscle size was reduced. Motoneurons formed both correct and incorrect connections not only in vitro when the pedal ganglion was attached to only one of two wing muscles, but also in whole-animal 'no choice' experiments when only one muscle was available for reinnervation. In these experiments incorrect connections were stable and were not eliminated at the later stages, as happened in experiments in which both muscles were accessible. In whole-animal experiments with reduced size muscles, a normal pattern of regeneration was conserved although not all incorrect connections were eliminated. Thus, in the course of regeneration: (i) locomotor motoneurons make connections with both correct and incorrect muscles; (ii) if for some group of motoneurons the correct targets are unavailable, the incorrect connections survive and become stable; (iii) if both groups of motoneurons have a choice between the correct and incorrect targets, initial mixed innervation is replaced by purely correct innervation; (iv) elimination of incorrect synapses could be a result of the competition between correct and incorrect synapses of the same neuron.
 
 

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa₃, which is a cytochrome c oxidase, and cytochrome aa₃ and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27 that lacks the aa₃ and caa₃ terminal oxidases and is also deficient in succinate: menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme 0. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase super-family. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b₅₅₈b₅₉₅b'.

The widely accepted model of reaction center /RC/ functioning is proved to come into contradiction with some recent data. in particular, it cannot explain why only a minor part of electronic excitations (~10%) escapes from excited RC special pairs hack to antenna BChls. Therefore we believe that the model must be substantially modernized. In 1981 we developed a new model /1,2/. We suggested a femtosecond stale to precede primary e-transfer reaction due to reorientation of water molecule dipole in the electric field of excited RC dimer. This mechanism is responsible for energy trapping before the primary e-transport occurs. During last years his mechanism got support from various experimental works. Now this polarization model claims to fit all reliable experimental data at least in bacterial photosynthesis.

The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN^- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b₅₉₅ and the low-spin heme b₅₅₈ components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b₅₅₈. At the same time, no evidence for significant binding of the ligands to the high-spin heme b₅₉₅ is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231—239] according to which the two high-spin hemes d and b₅₉₅ share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b₅₉₅.
 
 

Induction of the nonselective cyclosporin-sensitive pore in the inner mitochondrial membrane under conditions of complete dissipation of ion gradients and transmembrane potential was studied. This approach allows the kinetics of Ca²⁺-dependent pore opening and the preceding processes of induction to be studied separately. The effects of mitochondrial heterogeneity were also minimized. We found that the kinetics of pore opening can be described by a minimal two-step scheme where only the rate constant at the first step depends on Ca²⁺ concentration. Oxidation of pyridine nucleotides in the matrix caused a slow transition in the pore complex and decreased the apparent dissociation constant of the Ca²⁺-binding site from >1 mM to ~30 m M. N-Ethylmaleimide (but not disulfide-reducing agents) prevented and slowly reverted the pore induction process. Data suggesting allosteric modulation of the pore by pyridine nucleotides are presented.

Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of dicarboxylic (DC) acids at one side of BLM are monitored by measuring pH changes in the unstirred layers near the BLM surface via recording protonophore-dependent potentials. Two groups of DC acids are studied: (1) 2-n-alkylmalonic acids with an alkyl chain of different length which carry both carboxylic groups at one terminus of the hydrocarbon chain (a ,a -DC acids): and (2) dicarboxylic acids of different linear chain length having carboxylic groups at the opposite ends of the hydrocarbon chain (a ,w -DC acids). It is shown that the pH optimum of hydrogen ion fluxes for the DC acids is shifted considerably to acidic pH values compared to monocarboxylic acids and is located near pH 5. For both types of DC acids at pH << 5. the total transport is limited by diffusion of the anionic forms of the acids across the unstirred layers, while at pH >> 5 the transport is limited by diffusion of the neutral form across the membrane. The fluxes of a ,a -DC acids are similar to those of a ,w -DC acids provided that the acids have the similar number of carbon atoms, the fluxes grow with the increase in the chain length of the alkyl radical.
 
 

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q_B by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to Q_B (at 450 nm), (ii) the electrogenic proton delivery from the surface to the Q_B site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q_B^- and the proton-related electrogenesis proceeded with the same time constant of » 100 m s (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (t» 400 m s). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the Q_B site followed the reduction of Q_B without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.

Evidence is presented that the type strain of filamentous gliding bacterium Leucothrix mucor DSM 2157 is capable of lithoheterotrophic growth. Thiosulfate oxidation was accompanied by accumulation of sulfate and tetrathionate in the growth medium and intracellular accumulation of elemental sulfur as occurs in filamentous sulfur bacteria of the genus Thiothrix. Thiosulfate oxidation by L. mucor was induced during growth with a limiting concentration of organic substrate (i. e. 50-170 mg l^-1 tryptone) and resulted from the induction of dissimilatory enzymes of sulfur metabolism: Metabolically useful energy was derived by both substrate-linked and oxidative phosphorylalion from thiosulfate and sulfite oxidation. During sulfite oxidation the electron transfer pathway was shown to begin at the ubiquinone-cytochrome b segment of the electron transport chain. L. mucor limited by organic substrate had lowered levels of membrane bound cytochromes c and respiratory activity but a higher input of a cyanide-insensitive respiratory component than during organic enriched growth. Cytochromes aa₃ and d which had not been shown previously in this bacterium, were detected in the cells under various growth conditions. Cytochrome aa₃ is suggested to take part in the electron transfer pathway from thiosullate to oxygen, which was completely inhibited by 100 m M cyanide.
 
 

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz. glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-1DH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream"' activation by Ca²⁺, likely at the level of the F1FoATPase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ [Ca²⁺]_m which act as a signal to these activities. In this article, we review recent work in which [Ca²⁺]_m is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of [Ca²⁺]_m relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e. g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of [Ca²⁺]_m to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, streptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising [Ca²⁺]_m at a given, time-average value of cytosol tree Ca²⁺.
 
 

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncouples gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5-7 range. The efficiency of the long-lived 0 intermediate formation increases at a low pH. In the wild type as well as in the E204Q mutant purple membrane, the 0 intermediate decay is slowed down at slightly higher pH values than that of the purple-blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the 0 intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H⁺. At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the 0 intermediate. Under such conditions, membrane potential stabilization of the 0 intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.
 
 

Effect of a cationic polymer, poly (L-lysine), on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its negatively charged analogue 0-pyromellitylgramicidin (OPg) in a bilayer lipid membrane is studied using a method of sensitized photoinactivation. This newly developed method is based on the analysis of transmembrane current transients induced by a flash in the presence of a photosensitizer. It has been shown previously that the time course of the flash-induced current decrease in most cases follows a single exponential decay with an exponential factor (t , the characteristic time of photoinactivation) that correlates well with the single-channel lifetime. Addition of polylysine does not affect t for gA channels, but causes a substantial increase in t for OPg channels. This effect is reversed by addition of polyacrylic acid. The deceleration of the photoinactivation kinetics is ascribed to electrostatic interaction of polylysine with OPg probably resulting in OPg clustering. The latter can stabilize the channel state by reducing the rotational and lateral mobility of OPg monomers and dimers, and thus increase the single channel lifetime.
 
 

The generation of a transmembrane electric potential difference induced by a series of laser flashes was studied by the direct electrometrical method in proteoliposomes containing oxygen-evolving particles of photosystem II. In addition to the fast stage of generation of the membrane potential, which is due to electron transfer from the redox active tyrosine residue Tyr-161 (YZ) to the primary quinone acceptor QA, electrogenic stages corresponding to the S1 ® S2 (t = 30 m sec), S2 ® S3 (t = 240 m sec), and S3 ® S4 --> S0 (t = 6.2 msec) transitions of the oxygen-evolving complex (OEC) were observed. The amplitudes of the photoelectric responses show that the contribution of the OEC to the overall electrogenicity is small. The parameters of the electrogenic reactions of the OEC as measured in photosystem II preparations containing the peripheral proteins of 23 and 17 kD were similar to those of photosystem II preparations devoid of these peptides. It is concluded that neither the 23- nor the 17-kD proteins are involved in the electrogenic reactions of the OEC.

Effects of dicarboxylic fatty acids with varying positions of the carboxyl groups on respiration and membrane potential of liver mitochondria were studied. Tetradecylmalonic acid (a fatty acid with two carboxyl groups in the alpha-position) efficiently uncoupled oxidative phosphorylation similarly to palmitic acid with the same number of carbon atoms. Similarly to the uncoupling by palmitic acid, the coupling effects of carboxyatractylate and glutamate changed reciprocally with changes in pH of the incubation medium: on increasing the pH from 7.0 to 7.8, the coupling effect of carboxyatractylate increased and that of glutamate decreased. A dicarboxylic fatty acid with the second carboxyl at the end of the alkyl chain in the omega-position (a ,w -tetradecyldicarboxylic acid) stimulated respiration of the mitochondria at a significantly higher concentration than myristic acid with the same number of carbon atoms, but unlike the latter nearly failed to decrease the transmembrane potential D y . Neither carboxyatractylate nor glutamate inhibited the respiration stimulated by this dicarboxylic fatty acid.
 
 

We have demonstrated the temperature dependence of the steady-state fluorescence lineshape of the BChl c band measured for intact cells of the green bacterium Chloroflexus aurantiacus over the 1.8-293 K range. The measured temperature dependence has been shown to be in a good agreement with theoretical one, calculated for our original model of pigment organization in the chlorosomal oligomeric antenna of green photosynthetic bacteria based on spectral hole burning studies (Biophys.J., 1996, 71:995-110). This model implies that the BChl c antenna unit is a tubular aggregate of six exciton-coupled linear pigment chains having the exciton level structure with strongly allowed higher levels.
 
 

A model of pigment organization in the B808-866 bacteriochlorophyll a antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed. The building block of the antenna was assumed to be structurally similar to that of the B800-850 light-harvesting 2 (LH2) antenna of purple bacteria and to have the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers, where N = 24 or 32. We have shown that the O_y transition dipoles of BChl808 and BChl866 molecules form the angles 43° ± 3° and 8° ± 4°, respectively, with the plane of the corresponding rings. Using the exciton model, we have obtained a quantitative fit of the pump-probe spectra of the B866 and B808 bands. The anomalously high bleaching value of the B866 band with respect to the B808 monomeric band provided the direct evidence for a high degree of exciton delocalization in the BChl866 ring antenna. The coherence length of the steady-state exciton wave packet corresponds to five or six BChl866 molecules at room temperature.

In this work we explain the spectral heterogeneity of the absorption band (Monshouwer et al., 1995. Biochim. Biophys. Acta. 1229:373-380), as well as the spectral evolution of pump-probe spectra for membranes of Rhodopseudomonas (Rps.) viridis. We propose an exciton model for the LH1 antenna of Rps. viridis and assume that LH1 consists of 24-32 strongly coupled BChl b molecules that form a ring-like structure with a 12- or 16-fold symmetry. The orientations and pigment-pigment distances of the BChls were taken to be the same as for the LH2 complexes of BChl a-containing bacteria. The model gave an excellent fit to the experimental results. The amount of energetic disorder necessary to explain the results could be precisely estimated and gave a value of 440-545 cm^-1 (full width at half-maximum) at low temperature and 550-620 cm^-1 at room temperature. Within the context of the model we calculated the coherence length of the steady-state exciton wavepacket to correspond to a delocalization over 5-10 BChl molecules at low temperature and over 4-6 molecules at room temperature. Possible origins of the fast electronic dephasing and the observed long-lived vibrational coherence are discussed.
 
 

Recent structure determinations suggested a new binding site for a non-redox active metal ion in submit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paruroccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca²⁺ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme it visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca²⁺ ligand.
 
 

Glutaraldehyde, aluminum ions and glycerol (that inhibit the M intermediate decay in the wild-type bacteriorhodopsin and azide-induced M decay in the D96N mutant by stabilization of the M_closed) accelerate the N decay in the D96N mutant. The aluminum ions, the most potent activator of the N decay, induce a blue shift of the N difference spectrum by ~10 nm. Protonated azide as well as acetate and formate inhibit the N decay in both the D96N mutant and the wild-type protein. It is concluded that the N intermediate represents, in fact, an equilibrium mixture of the two ('open' and 'closed') forms. These two forms, like M_closed and M_opend come to equilibrium in the microsecond range. The absorption spectrum of the N_open is slightly shifted to red in comparison to that of the N_closed. Again, this resembles the M forms. l3-cis-all-trans re-isomerization is assumed to occur in the N_closed form only. Binding of 1-2 molecules of protonated azide stabilizes the N_open form. Existence of the 'open' and 'closed' forms of the M and N intermediates provides the appropriate explanation of the cooperative phenomenon as well as some other effects on the bacteriorhodopsin photocycle. Summarizing the available data, we suggest that M_open is identical to the M_N form, whereas M₁ and M₂ are different substates of M_closed.
 
 

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the MN intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the MN (M_open) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 ~ 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (~10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the MN (M_open) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate that observed at lower pH has the closed inward proton channel.
 
 

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 m M tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters’ function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.

The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength radiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependencies of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential.
 
 

ba₃-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a m s/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba₃ by RuBpy results in two phases of membrane potential generation by the enzyme with t values of about 20 and 300 m s at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba₃ reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H₂O₂ with a second order rate constant of ~2x10³ M^-1 s^-1. The data indicate that single electron reduction of ba₃ oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba₃, is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba₃ oxidase as compared to the oxidases of the aa₃-type.
 
 

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl—oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl—oxo-to-oxidized (F ® 0) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389-393]. As in the case of the F ® 0 transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a t of 40—50 m s (reduction of heme) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is ~4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F ® 0 electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both "chemical" and "pumped" protons. The protonic part of the P ® F electric response is faster than in the F ® 0 transition and can be deconvoluted into three exponential phases with t values varying for different samples in the range of 0.25—0.33, 1—1.5, and 6—7.5 ms al pH 8. Of these three phases, the 1 —1.5 ms component is the major one contributing 50—60%. The P ® F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P ® F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the cub redox state in controlling the rate of P ® F conversion of heme a₃ is discussed.
 
 

Physiological aspects of uncoupling of oxidation and phosphorylation are reviewed in the context of involvement of mitochondrial anion carriers. It is assumed that the carriers facilitate electrophoretic translation of fatty acid anion, RCOO^-, from the inner to the outer leaflet of the mitochondrial membrane, whereas back movement of the protonated fatty acid, RCOOH, from the outer to the inner leaflet represents flip-flop of RCOOH via the phospholipid bilayer of the membrane. The RCOO- transport seems to be catalyzed by the ATP/ADP and aspartate/ glutamate antiporters, dicarboxylate carrier, and uncoupling proteins (UCP1, UCP2, UCP3L, UCP3S, and plant UCP). The fatty acid uncoupling is shown to be involved in the thermoregulatory heat production in animals and plants exposed to cold, as well as in performance of respiratory functions other than ATP synthesis, i.e., formation of useful substances, decomposition of unwanted substances, and antioxidant defense. Moreover, partial uncoupling might take part in optimization of the rate of ATP synthesis in aerobic cells.
 
 

A decrease in the extracellular H⁺ concentration creates difficulties for membrane-linked energetics in bacteria employing H⁺ as the coupling ion. At high extracellular pH (pH_o), H⁺ ions pumped from the cell by, say, the respiratory chain, are immediately neutralized by the alkaline extracellular medium. Under such conditions, the only driving force that might compel outer H⁺ ions to return to cytosol and perform their function is the electric potential difference across the cytoplasmic membrane (Dy ). However, when D pH in the opposite direction is equal to, e.g., 2 pH units (intracellular pH = 7.5 at pH_o = 9.5), D y would be so high that the risk of membrane electric breakdown would increase. This is why some bacteria deal with high pH by, for example, replacing H⁺ by Na⁺ as the coupling ion rather than by increasing D y . It has been shown in several species of bacteria that the alkalinization of the growth medium induces primary Na⁺ pumps (e.g. Na⁺-motive respiratory chain enzymes and Na⁺ ATPase). Electrogenic Na⁺ efflux via these pumps produces an electrochemical Na⁺ potential difference (D m_Na⁺) composed of Dy and D pNa⁺. Dm _Na⁺ can be used to perform various types of membrane-linked work. The Dy constituent of Dm _Na⁺ may maintain electrophoretic influx of H⁺ such that the alkalinization of cytoplasm is prevented. The latter function may be supported by a mechanism based on the uphill influx of C1^- instead of Na⁺. This seems to be the case for alkaliphilic and halophilic Natronobacter pharaonis. There is an indication that not only Na⁺ but also Ca²⁺ may substitute for H⁺ in Gleobacter violaceus growing at high pH.
 
 

The review summarizes the present state of our knowledge concerning alternative functions of mitochondria, namely energy conservation in forms of protonic potential and ATP, thermoregulatory energy dissipation as heat, production of useful substances, decomposition of harmful substances, control of cellular processes. The recent progress in understanding of some mitochondrion-linked pathologies is described. The role of reactive oxygen species in these processes is stressed. Possible mechanisms of programmed death of mitochondrion (mitoptosis), cell (apoptosis) and organism (phenoptosis) are considered. A concept is put forward assuming that mitoptosis is involved in some types of apoptosis whereas apoptosis can be a part of a phenoptotic cascade. It is hypothesized that septic shock, as well as the stress-induced brain and heart ischemic diseases and cancer exemplify mechanisms of phenoptosis purifying population from dangerous or useless individuals.
 
 

As a strong oxidant, molecular oxygen, on the one hand, is favorable for cell energetics based on the oxidative degradation of respiratory substrates, but, on the other hand, is dangerous as a precursor of reactive oxygen species (ROS) that damage intracellular compounds including DNA. To minimize the ROS-induced damage, living organisms have created a distributed in depth multilane anti-ROS system. However, sometimes ROS appear to be biologically useful. ROS play a role of "biological weapon" (phagocytosis) and "bad message" (apoptosis), and perhaps components of putative mutator system which increases the genetic diversity of populations. The latter effect, obviously useful for biological evolution, might be unnecessary and even harmful for Homo sapiens who relies now on technical progress rather than evolution. Identification of (i) mutators, i. e. enzymes that generate ROS to induce mutagenesis and aging and (ii) inhibitors of mutator activity is proposed to be a new approach to the solution of the lifespan increase problem.
 
 

The concept formulated here presumes the existence of specific mechanisms of evolution that save intermediate (and therefore imperfect) forms of organisms from elimination by natural selection. A change in the life strategy made in situations when the appearance of a new trait worsens, rather than improves, adaptation of the organism to the changing environment can be one of these mechanisms. The concept postulates that, in such cases, K-strategy (relatively low rates of reproduction and activity in general but long life span) is replaced by r-strategy (high activity and reproduction but short life span). A decrease in the life span upon the K ® r transition is suggested to be an unavoidable consequence of an elevation of formation of toxic reactive oxygen species under conditions of increased rates of aerobic metabolism required for the increased life activity. The phenomenon of gigantism of transgenic tobacco plants that overproduce a mitochondrial heat shock protein (experiments done by A. Moore) is assumed to be explained by an r ® K transition. On the other hand, a decrease in the life activity and a considerable increase in life span occurring in a nematode upon mutations inhibiting the CoQ biosynthesis (S. Hekimi) might serve as an example of a K ® r transition.
 
 

The role of reactive oxygen species (ROS) participating in antiviral host defense is considered. Unlike antibacterial defense, when ROS and their derivatives act as biological weapons killing pathogenic bacteria, the function of ROS in the antiviral defense is assumed to be mediated by apoptosis. It is suggested that a cell activate superoxide generation and hydrogen peroxide by xanthine oxidase as well as by intracellular NADPH-oxidase in response to appearance of a virus in its cytoplasm. Increase in ROS level turns on the process of programmed cell death in the infected cells. Moreover, H₂O₂ diffuses into the adjacent cells (due to its high membrane permeability), also inducing apoptosis (death of bystander cells). So, the infected cell and its neighbors (which are the most likely to be infected) are eliminated, thus blocking the spreading of the viral infection.
 
 

Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10^-3 M) for 25 min apparently swell, increase their [Ca²⁺]_i, with obvious depolarization of the mitochondrial membrane. In 3 h after ouabain was omitted from the solution, 62 ± 3% of granule cells had pycnotic nuclei. The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosplionoheptanoate (10^-4 M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of [Ca²⁺]_i. These data suggest that cellular Na⁺/K⁺-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca²⁺ and Na⁺ influxes into the cells through the channels activated by Glu. This process leads to cell swelling, mitochondrial deenergization and death of granule cells. Possibly, the decrease of Na⁺/K⁺-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.

Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10^-3 M) for 25 min apparently swell, increase their [Ca²⁺]_i, with obvious depolarization of the mitochondrial membrane. In 3 h after ouabain was omitted from the solution, 62 ± 3% of granule cells had pycnotic nuclei. The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosplionoheptanoate (10^-4 M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of [Ca²⁺]_i. These data suggest that cellular Na⁺/K⁺-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca²⁺ and Na⁺ influxes into the cells through the channels activated by Glu. This process leads to cell swelling, mitochondrial deenergization and death of granule cells. Possibly, the decrease of Na⁺/K⁺-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.
 
 

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.

Magnesium-supported PP_i hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PP_i and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PP_i hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme-substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.
 
 

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg²⁺-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast. Mg²⁺-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg²⁺ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis. Mg²⁺ binds to 2.5 sites per monomer, supporting the notion that one of the tighter binding sites is located at the trimer-trimer interface. Mg²⁺ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg²⁺ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.
 
 

The binding of denatured B. stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E. coli chaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL. Similar apparent K_D values for the complex GroEL-GAPDH were obtained in both cases (0.04 and 0.03 m M, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer. Addition of GroEL and Mg× ATP to a reactivation mixture increased the yield of reactivation of both e. coli and B. stearothermophilus GAPDHs. Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction on a wild-type E. coli cell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form. These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.

The thioredoxin action upon the 2-oxoacid dehydrogenase complexes is investigated by using different thioredoxins, both wild-type and mutated. The attacking cysteine residue of thioredoxin is established to be essential for the thioredoxin-dependent activation of the complexes. Mutation of the buried cysteine residue to serine is not crucial for the activation, but prevents inhibition of the complexes, exhibited by the Clamydomonas reinhardtii thioredoxin m disulfide. Site-directed mutagenesis of D26, W31, F/W 12, and Y/A70 (the Escherichia coli thioredoxin numbering is employed for all the thioredoxins studied) indicates that both the active site and remote residues of thioredoxin are involved in its interplay with the 2-oxoacid dehydrogenase complexes. Sequences of 11 thioredoxin species tested biochemically are aligned. The thioredoxin residues at the contact between the a 3/3₁₀ and a 1 helices, the length of the a 1 helix and the charges in the a 2-b 3 and b 4-b 5 linkers are found to correlate with the protein influence on the 2-oxoacid dehydrogenase complexes (the secondary structural elements of thioredoxin are defined according to Eklund H et al., 1991, Proteins 11: 13-28). The distribution of the charges on the surface of the thioredoxin molecules is analyzed. The analysis reveals the species-specific polarization of the thioredoxin active site surroundings, which corresponds to the efficiency of the thioredoxin interplay with the 2-oxoacid dehydrogenase systems. The most effective mitochondrial thioredoxin is characterized by the strongest polarization of this area and the highest value of the electrostatic dipole vector of the molecule. Not only the magnitude, but also the orientation of the dipole vector show correlation with the thioredoxin action. The dipole direction is found to be significantly influenced by the charges of the residues 13/14, 51, and 83/85, which distinguish the activating and inhibiting thioredoxin disulfides.
 
 

The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of poly cation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme.
 
 

The nonstoichiometric polyelectrolyte complex (PEC) formed by poly (methacrylic acid) (degree of polymerization 1830) (PMAA) and poly (A'-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5-6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH-PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble. PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly.
 
 

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg²⁺ ions/enzyme hexamer in water-filled cavities formed by Asn²⁴, Ala²⁵, and Asp²⁶ at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg²⁺ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg²⁺ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg²⁺ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg²⁺ at pH 7.2 is intermediate between those of two Mg²⁺ binding sites found in the E-PPase active site. It is concluded that the metal ion-binding site that found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg²⁺ greatly stabilizes the enzyme hexamer but has little effect on catalysis.
 
 

The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium-ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH-synthase in the course of the reaction (k_in), (b) prostanoid metabolism (k_r), and (c) incorporation of exogenous AA into cellular membranes (k_a). From our experiments and mathematical calculation these parameters were found to be k_in= 0.20 ± 0.02 min^-1, k_r = 0.17 ± 0.03 min^-1 in the case of stimulation with A23187, and k_in = 0.0156 min^-1, k_r = 0.0134 min^-1, k_a = 0.0025 min^-1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of k_in=0.20 ± 0.02 min^-1. This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10-fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH-synthase only. A regulation mechanism based on the co-operation of the constitutive PGH-synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling.
 
 

The dynamics of prostaglandin (PG) E₂ synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE₂ synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE₂ synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE₂ metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE₂ synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE₂ consumption (k₂) and PGH-synthase inactivation in the course of the reaction (k_in) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated k_in value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE₂ synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent k_in and k₂ values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.
 
 

Monoclonal antibodies of two clones reacting with the nonnative forms of D-glyceraldehyde-3-phosphate dehydrogenase, EC 1. 2. 1. 12 (GAPDH), were obtained. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G7 belonged to IgM type. The interaction of antibodies of both clones with the immobilized and soluble enzyme was studied. The specificity of antibodies to the definite oligomeric forms was demonstrated on immobilized monomers, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The binding of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous renaturation of GAPDH. The immobilized antibodies of both clones were successfully used for the purification of GAPDH solution from the denatured admixtures.
 
 

Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD⁺-binding domain. At pH 8. 0, the transition temperatures (t_max) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3°C and 61.9°C, with calorimetric enthalpies (D H_cal) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively. In the presence of nearly saturating NAD⁺ concentrations, the t_max and the D H_cal increased by 13.6°C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8°C and 109 kJ/mol for the isolated domain. These results indicate that interdomain interactions are essential for NAD⁺ to produce its stabilizing effect on the structure of the native enzyme. The thermal stability of the isolated NAD⁺-binding domain increased considerably upon transition from pH 6.0 to 8.0. By contrast, native GAPDH exhibited greater stability at pH 6.0: similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli. The binding of NAD⁺ to rabbit muscle apoenzyme increased t_max and D H_cal and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations. Alkylation of the essential Cysl49 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD⁺. Replacement of Cysl49 by Ser or by Ala in the B. stearothermophilus GAPDH produced some stabilization, the effect of added NAD⁺ being basically similar to that observed with the wild-type enzyme. These data indicate that neither the ion pairing between Cysl49 and His 176 nor the charge transfer interaction between Cysl49 and NAD⁺ make any significant contribution to the stabilization of the enzyme's native tertiary structure and the accomplishment of NAD⁺-induced conformational changes. The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both D H_cal and t_max. Interestingly, NAD⁺ binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition.
 
 

 Inhibition of Select Mitochondrial Enzymes in PC 12 Cells Exposed to S- (l, l, 2, 2-Tetrafluoroethyl) -L-Cysteine

Many halogenated foreign compounds are detoxified by conversion to the corresponding cysteine S-conjugate, which is N-acetylated and excreted. However, several halogenated cysteine S-conjugates [e. g. S- (l, l, 2, 2-tetrafluoroethy) -L-cysteine (TFEC)] are converted to mitochondrial toxicants by cysteine S-conjugate b -lyases. In the present work, we showed that TFEC appreciably inactivated highly purified a -ketoglutarate dehydrogenase complex (KGDHC) in the presence of a cysteine S-conjugate b -lyase. Incubation of PC12 cells (which contain endogenous cysteine S-conjugate b -lyase activity) with TFEC led to a concentration and time-dependent loss of endogenous KGDHC activity. A 24-hr exposure to 1 mM TFEC decreased KGDHC activity in the cells by 90%. Although treatment with TFEC did not inhibit intrinsic pyruvate dehydrogenase complex (PDHC) activity, it inhibited dichloroacetate/Mg²⁺-mediated activation/dephosphorylation of PDHC in the PC12 cells by 90%. To determine the selectivity of enzymes targeted by TFEC, several cytosolic and mitochondrial enzymes involved in energy metabolism [malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, cytosolic and mitochondrial aspartate amino-transferases (AspAT)] were also assayed in the PC12 cells exposed to 1 mM TFEC for 24 hr. Of these enzymes, only mitochondrial AspAT, a key enzyme of the malate-aspartare shuttle, was inhibited. The present results demonstrate a selective vulnerability of mitochondrial enzymes to toxic cysteine S-conjugates. The data indicate that TFEC may be a useful cellular/mitochondrial toxicant for elucidating the consequences of the diminished mitochondrial function that accompanies numerous neurodegenerative diseases.
 
 

A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His¹³⁶ and His¹⁴⁰ in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytia, T., Goldman, A-, Coopennan, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr⁷⁷ and Gln⁸⁰ in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg²⁺ concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by l0⁶ fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg^2+-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr⁷⁷, Gln⁸⁰, and His¹³⁶ exhibits positive cooperativity in catalysis, consistent with this model.

The hydrogen peroxide-induced 'non-phosphorylating' activity of D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown to be a result of the successive action of two forms of the enzyme subunits: one catalyzing production of 1, 3-bisphosphoglycerate, and the other performing its hydrolytic decomposition. The latter form is produced by mild oxidation of GAPDH in the presence of a low hydrogen peroxide concentration when essential Cys-149 is oxidized to the sulfenate derivative. The results obtained with a C153S mutant of Bacillus stearothermophilus GAPDH rule out the possibility that intrasubunit àñó I transfer between Cys-149 and a sulfenic form of Cys-153 is required for the 'non-phosphorylating' activity of the enzyme.
 
 

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokarvotic pyrophosphatases.
 
 

Stability and catalytic properties of native and immobilized penicillin acylase were studied in systems with low water content. Preparations of both native and immobilized penicillin acylase demonstrated the catalytic activity even in solid-phase systems which contained 3-5 wt. % of water. The stability and catalytic activity of penicillin acylase at low water content depended on the thermodynamic water activity (aw) in the system.
 
 

Alternative states of the predicted structure of hairpin 17 in the small-subunit ribosomal RNA genes are described. Each of these structures is characteristic of one or several phylons of animals. For example, one of the alternatives could be found in the rRNA of most Bilateria, but not in protists, fungi, plants, or diploblastic animals. A presumable secondary structure of the 18S rRNA hairpin 17 of a lower multicellular animal, Rhopalura ophiocomae (Mesozoa: Orthonectida), was constructed. It differs drastically from the one of other lower multicellular animals, i.e., Rhombozoa, Myxozoa, and Acoela. The evolution of rRNA fragments according to the principle of punctuated equilibrium is discussed.
 
 

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10-min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca²⁺ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (r  = 1.718 g/cm³) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.
 
 

HIV-1 DNA integration is carried out by integrase, a viral protein which binds to specific sequences located on both extremities of the HIV-1 DNA LTR. Inhibition of integration was observed with submicromolar concentrations of mono- or bi-functionalized 11-mer oligonucleotide-intercalators, which were designed to form an alternate strand triple helix with the U5 LTR end containing two adjacent purine tracts on opposite strands 5'-GGAAAATCTCT-3'/3'-CCTTTTAGAGA-5'.

Integration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3'-end processing and strand transfer. A 3'-3' branched oligonucleotide functionalised by the intercalator oxazolopyr-idocarbazole at each 5'-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition.
 
 

We sequenced the promoter and the 5' region of the 19S rRNA gene of Astasia longa (Flagellata) and mapped the 5' end of the gene with reverse transcriptase. The gene proved highly homologous to its Euglena gracilis var. bacillaris counterpart, suggesting close phylogenetic relations of these species. Sequences conserved among Flagellata and Saccharomyces cerevisiae were found in the 5'-region of the gene. The promoter region upstream of the A. longa 19S rRNA gene was shown to contain a repetitive sequence which is characteristic of gene promoters of Giardia lamblia (Flagellata) and is possibly involved in transcription regulation.

The formation of germinal centers (GCs) represents a crucial step in the humoral immune response. Recent studies using gene-targeted mice have revealed that the cytokines tumor necrosis factor (TNF), lymphotoxin (LT) a , and LTb , as well as their receptors TNF receptor p55 (TNFRp55) and LTb R play essential roles in the development of GCs. To establish in which cell types expression of LTb R, LTb , and TNF is required for GC formation, LTb R^-/-, LTb^-/-, TNF^-/-. B cell-deficient (BCR^-/-), and wild-type mice were used to generate reciprocal or mixed bone marrow (BM) chimeric mice. GCs, herein defined as peanut aggluti-nin-binding (PNA⁺) clusters of centroblasts/centrocytes in association with follicular dendritic cell (FDC) networks, were not detectable in LTb R^-/- hosts after transfer of wild-type BM. In contrast, the GC reaction was restored in LT^-/- hosts reconstituted with either wild type or LTb R^-/- BM. In BCR^-/- recipients reconstituted with compound LTb^-/-/BCR^-/- or TMF^-/-/BCR^-/- BM grafts, PNA⁺ cell clusters formed in splenic follicles, but associated FDC networks were strongly reduced or absent. Thus, development of splenic FDC networks depends on expression of LTb and TNF by B lymphocytes and LTb R by radioresistant stromal cells.
 
 

A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study or primates and lizards. It was shown that electrophoretic bands or DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeals. Another group represents variable taxonprint bands chat differs even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated chat this approach could be of general utility for molecular systematics and species identification.
 
 

The ability of oligodeoxyxylopyrimidilates to form triplexes with complementary single-stranded DNA at the neutral pH was found. The complex composition, relative strand orientation and base-pairing scheme were determined using electromobility shift assay and thermal denaturation experiments.
 
 

This investigation is devoted to design of short "switch" oligonucleotides mono- or bi-functionnalized with intercalating agents capable to form a stable triplex with HIV integrase-cognate sequences and inhibit selectively HIV integration. Methods of intercalator incorporation at 5'- and/or 3'-terminal positions or one of the pyrimidine heterocyclic bases are developed.
 
 

The influence of daily fractional irradiation of male Wistar rats for 30 days on DNA-protein cross-links (DPC) in spleen, thymus, and liver cells was studied. The level of DPC depended strongly on the daily dose of irradiation and the studied organ. After irradiation at dose 0.5 Gy per day increased DPC level was detected in all organs. The highest level was in the lymphoid organs and the lowest in the liver. After irradiation at dose 0.3 Gy per day DPC formation was detected only in the thymus. The data suggest the existence of a dose threshold for DPC formation during fractional irradiation.

We developed a rapid screening system, using two reporter genes under the control of the same promoter, to identify the biological activity of modified or/and vectorized antisense oligodeoxynucleotides (ODNs). The ability of a dendrimeric structure and a monocationic cholesterol derivative to enhance ODN cellular uptake was previously investigated by fluorescence analysis. Then, the assay system was validated through investigating the effect of both vectors on antisense ODN efficiency.
 
 

We studied the uptake and intracellular distribution of an FITC labeled phosphodiester oligodeoxynucleotide (ODN) vectorized by a dendrimeric structure in cell culture.
 
 

Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents arc inefficient cellular uptake and intracellular transport to RNA target. To overcome these obstacles, ODN carriers have been developed, hut the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization. Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically. These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness. We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase. In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa. This system was validated through investigating the effect of the dendrimer vector on ODN biological activity. Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.
 
 

Mice deficient in lymphotoxin (LT)-a lack peripheral lymph nodes and Peyer's patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTa is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTa for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTa -deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTa^-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTa^-/- mice. Although LTa^-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTa ^-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTa signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.

The lichen family Umbilicariaceae is accepted by most lichenologists as consisting of two genera, Lasallia and Umbilicaria. The monophyly of these two genera was examined by phylogenetic analyses of nucleotide sequences of ITS1 and ITS2 rDNA. Sequences of these regions from three Lasallia and 17 Umbilicaria species were aligned to those of seven representatives of the outgroup taxa including Eurotiales, Onygenales and Caliciales (Mycocaliciaceae) and subjected to maximum parsimony, maximum likelihood and neighbour joining analyses. The resulting phylogenetic hypotheses supported the monophyly of the representative species of Lasallia. However, the species of Umbilicaria did not form a monophyletic sister-group to Lasallia due to the basal placement of other Umbilicaria species in some analyses. Based on these analyses, if Lasallia is recognized as a separate genus then Umbilicaria appears to be paraphyletic. Although further taxon sampling is required to resolve the monophyly of Umbilicaria, for the present we recommend retaining the current treatment of Lasallia as separate from Umbilicaria.

To elucidate the mechanism of interaction of restriction endonuclease fcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease fcoRII, form two types of stable complexes on native gel in the absence of Mg²⁺ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg²⁺. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of fcoRII endonuclease action is presented.
 
 

Evolutionary relationships among 116 representatives (80 genera) of Apiaceae (Umbelliferae) subfam. Apioideae were investigated by comparative sequencing of the two internal transcribed spacers of the 18S-26S nuclear ribosomal DNA repeat. The resultant phylogenies inferred using maximum parsimony and neighbor-joining methods clarified the relationships of several genera whose phylogenetic placements have heretofore been problematic. Comparisons between the phylogenies inferred and the distribution of several phytochemical (coumarins, flavonoids, and phenylpropenes) and morphological (stomates, pollen, and cotyledonary shape) characters were also made, revealing that many of these characters (like those morphological and anatomical characters of the fruit) are highly homoplastic. It is not surprising then that systems of classification of Apioideae based on these characters, particularly with regard to tribal and subtribal designations and relationships, are unsatisfactory. The results of recent serological investigations of the subfamily support several relationships proposed herein using molecular data.
 
 

A fraction of unusual fast sedimenting (10 min at 600-1700g) particles with properties of mitochondria has been detected in wheat seedlings. This fraction conventionally called "heavy" mitochondria amounts (by protein) to about 40% of the total subcellular particle fraction sedimented by 10-min centrifugation at 17,000g. The specific feature of these "heavy" mitochondria in aging tissues is an ability to synthesize and even superproduce heavy (r = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). The share of "heavy" mitochondria sedimented in the interval between 1000 and 1700g and possessing the maximal H-mtDNA synthesis in aging coleoptiles is about 1.5-fold higher than that in young coleoptiles. Although "heavy" mitochondria are present in young plant organs, they seem to be unable to synthesize H-mtDNA; heavy mtDNA forms only in mitochondria of aging or old cells. Thus, aging in plants is accompanied by a change in population of mitochondria and appearance of the ability for selective H-mtDNA superproduction in a certain mitochondrial fraction. Mitochondria isolated from wheat coleoptiles are practically not stimulated by uncouplers. "Heavy" (600-1700g) and usual (4,300-17,400g) mitochondria are similar in respiration rates, cytochrome compositions, cytochrome c amount (per mg protein) and sensitivities to respiration inhibitors. However, "heavy" mitochondria contain (per mg protein) cytochromes b and aa₃ by 10-20% and Ca²⁺ by 2-3-fold more than normal mitochondria. Ultrastructural analysis showed that the isolated fraction of fast sedimenting mitochondria consists of a suspension of closed membrane vesicles filled with cytoplasm and containing one or a few mitochondria. We observed similar structures in situ in vacuoles of parenchyma cells in the apical part of intact coleoptiles. The process of formation of such structures was detected by serial ultra-thin section analysis. It was shown that tonoplast protrudes into vacuoles, the separate mitochondria translocate into these protrusions, and then these structures separate. As a result, the suspended cytoplasmic bodies containing mitochondria appear in vacuoles. Appearance of these bodies containing mitochondria and, in particular, the superproduction of H-mtDNA in them correlate with processes of aging and cell transition to apoptosis.
 
 

Internucleosomal fragmentation of nuclear DNA (nDNA) [(180 ± 10 base pairs)_n, n ³ 1], characteristic of apoptosis, was observed in leaf and coleoptile zones of wheat (Triticum aestivum L.) seedlings, and labeled precursors were found to incorporate rapidly into the "heavy" (r = 1.716 g/cm3) fraction of mitochondrial DNA (hmtDNA). When separated by electrophoresis in agarose, the fragments of hmtDNA differed by alues proportional to 195 ± 10 base pairs and were similar in this aspect to oligonucleosomal fragments produced by apoptic degradation of nuclear DNA. The amount of hmtDNA in older (over 190 h) coleoptiles of wheat seedlings was comparable to the amount of nDNA (r = 1.700 g/cm3) and reached as high as 50% of the total coleoptile DNA content. In younger (under 144 h) coleoptile, hmtDNA was not detected in CsCl gradients by UV absorption and could be discerned only by its radioactivity. In contrast to nDNA, hmtDNA did not contain 5-methylcytosine and was produced due to unusually active DNA synthesis in the mitochondrial fraction. The known inhibitors of DNA synthesis, cycloheximide and ethidium bromide, did not hamper superproduction of hmtDNA; on the contrary, these inhibitors notably stimulated hmtDNA synthesis. The signal for hmtDNA synthesis and accumulation arrived in the course of the fifth cycle of synchronous DNA replication in nondividing leaf and coleoptile cells of 135–145-h-old seedlings and apparently did not migrate further along the seedling. hmtDNA superproduction is a characteristic programmed event, which is synchronously developed in the senescing cells of intact plant organs. The emergence and accumulation of hmtDNA can be used as an indicator of senescence.
 
 

The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2'deozyuridine-5'-triphosphate was synthesized via acylation of 5-aminoallyl-2'-deoxyuridine-5'-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.
 
 

Background: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to which are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell.

Results: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded b sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel b sandwich having some resemblance to 'jelly-roll' viral capsid protein structures.

Conclusions: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.

Inactivatio of genes encoding members of TNF and TNF receptor families reveals their divergent roles in the formation and function of secondary lymphoid organs. Most lymphotoxin a (lta )- and all lymphotoxin b receptor (ltb r)-deficient mice are completely devoid of lymph nodes (LNs); however, most lymphotoxin b (ltb )-deficient mice develop mesenteric LNs. Tnf- and tnfrp55-deficient mice develop a complete set of LNs, while ltb /tnfrp55 double-deficient mice lack all LNs, demonstrating cooperation between LTb and TNFRp55 in LN development. Now we report that ltb /tnfrp55 double-deficient mice develop the same set of mucosal LNs as do ltb -deficient mice, suggesting that ligands other than TNF signal through TNFRp55 during LN development. These LNs retain distinct T and B cells areas; however, they lack follicular dendritic cell networks. Structures resembling germinal centers can be found in the LNs from immunized ltb -deficient mice but not in ltb /tnf double-deficient mice. Additionally, stromal components of the spleen and LNs appears to be more severely disturbed in ltb /tnf double-deficient mice as compared with ltb -deficient mice. We conclude that LTb and TNF cooperate in the establishment of the correct microarchitecture of lymphoid organs.

Transcription of the TNF gene is rapidly and transiently induced by LPS in cells of monocyte/macrophage lineage. Previous data suggested that multiple NF-kB/Rel binding sites play a role in the transcriptional response to LPS of the murine gene. However, the relevance of homologous sites in the human TNF gene remained a matter of controversy, partly because the high affinity NF-kB/Rel site located at —510 in the murine promoter is not conserved in humans. Here we used two sets of similarly designed human and mouse TNF promoter deletion constructs and overexpression of Ikb in the murine macrophage cell line ANA-1 to show remarkable similarity in the pattern of the transcriptional response to LPS, further demonstrating the functional role of the distal promoter region located between —600 and —650. This region was characterized by mutagenesis of protein binding sites, including two relatively low affinity NF-kB/Rel sites, #2 and 2a. Mutation in each of the NF-kB sites resulted in 2- to 3-fold lower transcriptional activity in response to LPS. In contrast to LPS activation, the response to PMA was substantially lower in magnitude and required only the proximal promoter region. In summary, the functional topography of human and murine promoters when assayed in the same system has some marked similarities. Our observations support the notion that full LPS response of TNF gene requires both nf-kb and non-NF-kB nuclear proteins. Our data also suggest that the functional activity of a given kb site depends on the entire DNA sequence context in the promoter region.
 
 

A covalent complex formed by bacterial tRNAs and prothymosin a , an abundant acidic nuclear protein involved in proliferation of mammalian cells, upon production of the recombinant rat protein in Escherichia coli cells was studied. Several tRNA attachment sites were identified in the prothymosin a molecule using a combination of deletion analysis of prothymosin a and site-specific fragmentation of the protein moiety of the prothymosin a -tRNA complex. The electrophoretic mobilities of the tRNA-linked prothymosin a and its derivatives are consistent with one tRNA molecule attached to one prothymosin a molecule, thus suggesting that alternative tRNA linking to one of several available attachment sites occurs. The possible effect of tRNA attachment on the nuclear uptake of prothymosin a is discussed.
 
 

There is a vast body of literature on the quality control of protein folding and assembly into multisubunit complexes. Such control takes place everywhere in the cell. The correcting mechanisms involve cytosolic and organellar proteases: the result of such control is individual molecules with proper structure and individual complexes both with proper stoichiometry and proper structure. Obviously, the formation of organelles as such requires some additional criteria of correctness and some new mechanisms of their implementation. It is proposed in this article that the ability to carry out an integral (key) function may serve as a criterion of correct organelle assembly and that autophagy can be accepted as a mechanism eliminating the assembly mistakes.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.
 
 

Atlantic salmons, as well as Pacific salmons and trouts, were studied using electrophoretic analysis of nuclear DNA treated with short-splitting restriction endonuclease (taxonoprint). The DNA patterns of the species investigated can be clearly divided into four groups: (1) Salmo salar (Atlantic salmon); (2) Salmo trutta (trout), S. trutta caspius (Caspian trout), and S. ischchan (Sevan trout); (3) Parasalmo mykiss (Kamchatka steel-head), P. clarki (Clark’s salmon), and Kamchatka forms; (4) Oncorhynchus species (salmon trout O. masou, chum salmon O.keta, pink salmon O. gorbuscha, sockeyed salmon O. nerka, king salmon O. tschawycha, and coho salmon O. kisutch). It is suggested that these natural groups can be considered as taxons of the genus rank.
 
 

Fractional whole-body gamma-irradiation of mice at total doses of 0.5-1.5 Gy induces increased DNA-protein cross-links (DPCs) in thymus, spleen, and brain, whereas in liver no DPCs are detected. Chronic administration of zinc ions in drinking water at concentration 10 mg/liter for 20-30 days increased DPCs in thymus, spleen, brain, and liver of mice. The combined action of zinc ions and gamma-radiation produced a significantly lower amount of DPCs than was induced by the separate action of these agents.

Komarovia (Umbelliferae), a little known Middle-Asiatic monotypic genus of uncertain taxonomic relationships, has been compared with different Apioideae genera by means of morphological. immunochemical and nuclear ITS rDNA sequencing studies. The hypotheses of Komarovia affinity to Pyramidoptereae (Korovin, 1939) and Peucedaneae (Schischkin. 1951) have not been confirmed. Parasilaus has been shown as a genus closest to Komarovia.
 
 

Partial sequences of the rpoC1 gene from two species of angiosperms and three species of gymnosperms (8330 base pairs) were determined and compared. The data obtained support the hypothesis that angiosperms and gymnosperms are monophyletic and none of the recent groups of the latter is sister to angiosperms.
 
 

When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pimlp protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pimlp, respectively. These data imply that chaperones and Pimlp protease prefer their natural targets in mitochondria to imported foreign proteins. When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pimlp protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pimlp, respectively. These data imply that chaperones and Pimlp protease prefer their natural targets in mitochondria to imported foreign proteins.

A cryochemical synthesis of silver nanoparticles stabilized by poly(2-dimethylaminoethyl methacrylate) (poly-DMAEMA) has been performed; it has been found using optical spectroscopy, dynamic light scattering and electron microscopy that silver sols prepared with these nanoparticles in water, acetone and toluene are sterically stabilized by macromolecular poly-DMAEMA layers formed at the surface of nanoparticles, and the thickness of these layers depends on the nature of the solvent.
 
 

The effects of natural (a -tocopherol and ascorbic acid) and synthetic (3,5-dibutyl-4-hydroxytoluene, BHT) antioxidants added to growth medium on the callus formation, plant regeneration in a tissue culture of tomato plants, and growth of intact wheat seedlings was studied. The number and length of roots in regenerants from tomato cotyledons increased in the presence of a -tocopherol (2.7–27 mg/l). Ascorbic acid (10 mg/l) induced an increase in the mass of calluses originated from hypocotyl but inhibited root formation in regenerants from tomato cotyledons at 25°C. In contrast, it strongly stimulated root growth and formation at 15°C. BHT at 1.3 mg/l significantly enhanced the development of regenerants, but at 60 mg/l it strongly suppressed their development. BHT at 50 mg/l strongly inhibited the growth of etiolated wheat seedlings but slowed senescence of coleoptiles and induced synthesis of pigments (most probably, carotenoids) that accumulated mainly in roots. Thus, antioxidants, including BHT, effectively regulate in vitro and in vivo plant growth and development. Similar to other known regulators of plant growth, antioxidants may exert different and even opposite effects depending on their concentration in a medium and (for natural antioxidants) their explant origin and cultivation conditions (temperature).
 
 

Two nucleopeptides (NPs) were synthesized on the base of d -ornithine peptides by modification of the a -amino ornithine functions with pyrimidyl-1- and purinyl-9-acetic acids or with pyrimidyl-1- and purinyl-9-alanines. NPs were prepared on solid polymer bearing photolinker. Conjugates with the 16-mer oligonucleotide complementary to the env AUG codon region of the Friend murine leukemia virus were prepared.
 
 

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

A wealth of experimental data on the mechanism of the picornavirus genome replication has accumulated. Not infrequently, however, conclusions derived from these data appear to contradict each other. On the one hand, initiation of a complementary RNA strand can be demonstrated to occur in a solution containing only the poliovirus RNA polymerase, VPg, uridine triphosphate, poly(A) template and appropriate ions. On the other hand, convincing experiments suggest that efficient initiation of a viral complementary RNA strand require complex cis-acting signals on the viral RNA template, additional viral and possibly cellular proteins as well as a membrane-containing environment. On the one hand, there is evidence that the viral RNA, in order to be replicated, should first be translated, but on the other hand, the viral RNA polymerase appears to be unable to overcome the ribosome barrier. Possible solutions for these and several other similar paradoxes are discussed, along with less contradictory results on the properties of the picornaviral replicative proteins. Recent results suggesting that recombination and other rearrangements of the viral RNA genomes may be accomplished not only by the replicative template switching but also by nonreplicative mechanisms are also briefly reviewed.
 
 

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given including (i) double infections with movement-deficient (dependent) and helper viruses: (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus: and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and S'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.
 
 

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid control persistence by allowing the virus to infect cells in the white matter of the spinal cord.
 
 

Previously we showed that the ribonucleoprotein complexes (RNPs) of the TMV 30-kDa movement protein (MP) with TMV RNA are nontranslatable in vitro and noninfectious to protoplasts, but are infectious to intact plants. It has been suggested that MP-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata (Karpova et al., 1997, Virology 230, 11-21). The role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. Phosphorylation of the TMV MP, either before or after RNP complex formation, caused a conversion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.

The M1 protein is commonly believed to form a protein matrix underlying the lipid membrane of the influenza virus. Upon bombardment of virus particles with thermally activated tritium atoms (tritium planigraphy), the surface glycoprotein hemagglutinin and the matrix protein M1 were labeled, but not the nucleocapsid protein. Virion integrity proved to be the same in virus specimens before and after bombardment. Data on the M1 shielding in the virion and the flux attenuation coefficient of the lipid bilayer obtained with liposomes showed that the M1 matrix protein is embedded in the inner leaflet of the lipid membrane at the distance of 2.5¸ 5 nm from the outer virion surface.
 
 

To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PIAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PIAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.
 
 

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD₅₀). Mutants with similar PD₅₀s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.
 
 

Intact influenza A virions were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the matrix protein Ml was analyzed to determine the in situ accessibility of its tryptic fragments. These data were combined with the previously reported x-ray crystal structure of the Ml fragment 2-158 [Sha, B. & Luo, M. (1997) Nat. Struct. Biol. 4, 239-244] and the predicted topology of the C domain (159-252) to propose a model of MI arrangement in the virus particle.

Previously we reported that, unlike RNA of typical tobamoviruses, the translation of the coat protein (CP) gene of a crucifer-infecting tobamovirus (crTMV) in vitro occurred by an internal ribosome entry mechanism mediated by the 148-nt region that contained an internal ribosome entry site (IRES_CP,148^CR). The equivalent 148-nt sequence from TMV U1 RNA (U1_{CP, 148SP}) was incapable of promoting internal initiation. In the present work, we have found that the 228-nt region upstream of the movement protein (MP) gene of crTMV RNA (IRES_MP,228^CR) contained an IRES element that directed in vitro translation of the 3'-proximal reporter genes from chimeric dicistronic transcripts. Surprisingly, the equivalent 228-nt sequence upstream from the MP gene of TMV U1 directed translation of the downstream gene of a dicistronic transcript as well. Consequently this sequence was termed IRES_MP, ₂₂₈U1. It was shown that IRES_MP,₂₂₈CR, IRES_MP,₂₂₈U1, and IRES_CP,₁₄₈CR could mediate expression of the 3'-proximal GUS gene from dicistronic 35S promoter-based constructs in vivo in experiments on transfection of tobacco protoplasts and particle bombardment of Nicotiana benthamiana leaves. The results indicated that an IRES element was located within the 75-nt region upstream of MP gene (IRES_MP, ₇₅), which corresponded closely to the length of the 5'UTR of TMV subgenomic RNA (sgRNA) I2. The RNA transcripts structurally equivalent to I2 sgRNAs of TMV U1 and crTMV, but containing a hairpin structure (H) immediately upstream of IRES_MP,₇₅ (HIRES_MP,₇₅^CR-MP-CP-3’UTR; hires_mp,₇₅^u1-mp-cp-3’utr); were able to express the MP gene in vitro. The capacity of HIRES_MP, ₇₅^CR sequence for mediating internal translation of the 3'-proximal GUS gene in vivo, in tobacco protoplasts, was demonstrated. We suggested that expression of the MP gene from I2 sgRNAs might proceed via internal ribosome entry pathway mediated by IRES_MP element contained in the 75-nt 5'UTR. Our results admit that a ribosome scanning mechanism of the MP gene expression from I₂ sgRNA operates concurrently.
 
 

The barley stripe mosaic virus (BSMV) triple gene block (TGB) coding for movement proteins (MPs) was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. In particular, the BSMV/PSLV hybrid infected Nicotiana benthamiana, a nonhost plant for PSLV, indicating a contribution of non-MP elements of BSMV genome to host specificity of virus transport. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (b b gene) indicated that (i) sequence-independent binding of b b to viral RNAs could occur during formation of b b-RNA complexes in vivo and that (ii) the b b MP is involved in virus long-distance movement, for which homologous N- and C-terminal b b domains are required.
 
 

The origin of replication (oriR) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure that is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterized the kissing interaction. Site-directed mutations, which affected the different base pairs involved in the kissing interaction, were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base, which gaps the major groove of the kissing domain, was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.
 
 

Filamentous particles of beet yellows closterovirus (BYV) are built of two related capsid proteins, of which the minor species, p24, forms a 75 nm tail at one end of the virion. In the present work, we used polyclonal antibodies against p24 for isolating the 'tailed' virion segments from sonicated BYV particle preparations. The [g =³²P]ATP-labelled RNA obtained from the antibody-selected particle segments consistently showed stronger hebridization with the 5'-terminal BYV cDNA clones than with the 3'-terminal cDNA clones. These data clearly indicate that it is the 5'-terminal portion of the closterovirus RNA genome that is encapsidated by p24.