BIOENERGETICS AND PHOTOSYNTHESIS
GENE STRUCTURE, REGULATION AND EVOLUTION
CELL BIOLOGY AND PHYSIOLOGY
GENEBEE-SERVER OF RUSSIAN EMBnet NODE
LIST OF MAJOR PUBLICATIONS
Possible involvement of ATP/ADP antiporter and uncoupling protein
(UCP) in thermoregulatory uncoupling of oxidative phosphorilation in heart
muscle has been studied. To this end, effects of carboxyatractylate (cAtr)
and GDP, specific inhibitors of the antiporter and UCP, on the membrane
potential of the oligomycin-treated mitochondria from cold exposed (6oC,
48h) and control rats have been measured. It is found that cAtr increases
the membrane potential level in both cold-exposed and non-exposed groups,
the effect being strongly enhanced by cooling. As for GDP, it is effective
only in mitochondria from the cold-exposed rats. In these mitochondria,
the coupling effect of GDP is smaller than that of cAtr. GDP, which does
not interact with UCP, is without any influence on membrane potential.
The cold exposure is found to increase the uncoupling efficiency of added
natural (palmitate) or artificial (SF6847) uncouplers, the increase being
cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine
serum albumin enhances D y in both cold-exposed and control groups, the
effect being much larger in the former case. It is concluded that in heart
muscle mitochondria the ATP/ADP antiporter is responsible for the ‘mild
uncoupling’ under normal conditions and for major portion of the thermoregulatory
uncoupling in the cold whereas the rest of thermoregulatory uncoupling
is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown
to increase strongly in rat heart muscle under the cold exposure conditions
Light-driven ATP synthesis was found in the cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin and lacking the H+-pumping bacteriorhodopsin. Photophosphorylation occurred in the cells with the cyanide-inhibited respiratory chain as well as in the untreated cells at a low chloride concentration in the incubation medium. A rise in the chloride concentration from 0.1 to 2.35 M in the incubation medium led to the inhibition of photophosphorylation. Continuous illumination increased membrane D Y when the respiration was inhibited by cyanide. DCCD, an ATPase inhibitor, stimulated this effect. To explain the data, one may suggest that halorhodopsin pumped chloride into the cells whereas chloride efflux from the cells through Cl--ATP-synthase was coupled to the ATP synthesis (chloride cycle).
Under fully reduced conditions, reassociation kinetics of CO were
studied in several terminal oxidases containing copper in their binuclear
center. The purified Paracoccus denitrificans ba3-type
quinol oxidase was found to recombine with CO monophasically (t 25-30 ms)
like the H+-translocating oxidases of bo-type from
Escherichia coli, caa3-type from Bacillus halodurans
FTU, and bo-type from Methylobacillus flagellatum KT. H+-translocating
oxidase of the aa3-type from bovine heart recombined
with CO monophasically at a higher rate (t 16-19 ms) than the studied copper-containing
bacterial oxidases. After prolonged incubation in the presence of CO, oxidases
of ba3- and aa3-type changed their
CO-binding properties. The contribution of the slow component was diminished
while new fast components arose. Measurement of the metal content in the
oxidases indicated that during the incubation, the enzymes lost their copper,
the process being accompanied by the appearance of a fast CO recombination
rate resembling that of the non-copper, presumably Na+-translocating
oxidases of bd-type from E. coli and bb-type from
B. halodurans FTU. This points to a role of copper in CO-binding
by terminal oxidases.
The study on the H+/e- ratio in B.halodurans FTU cells showed that it was about zero when the bb-type oxidase dominated in the cells. When the H+/e- ratio was studied in the cells by the oxygen pulse method in the presence of protonophorous compound CCCP, an alkalization of the incubation medium was shown to take place. The alkalization decreased in the presence of thiocyanate (the compound decreasing D Y ) and was absent in the Na+-free medium. These data support the "Na+-cycle" hypothesis and suggest that a functional Na+-pump operates in the bacterium.
It was found that two NADH:ubiquinone oxidoreductases operate in the A.vinelandii respiratory chain: proton-motive NADH dehydrogenase (NDH-I) and enzyme non-coupled to the energy conservation (NDH-II). The level of these oxidoreductases strongly depends upon [O2] and [NH3] in the growth medium. Increase in [O2] results in lowering of the coupled enzyme level and in rise of the non-coupled one. Exclusion of NH3 from the growth medium increases the level of the non-coupled enzyme whereas that of the coupled enzyme remains constant.
It was found that A.vinelandii bd-type oxidase can produce D pH and D Y . The H+/e- ratio for this enzyme was established as being close to 1. It is concluded that the bd-type oxidase of A.vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase. It was also shown that Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A.vinelandii respiratory chain and does not in the bd-type quinoloxidase terminated branch.
Summarising the above observations one can assume that the respiratory protection of nitrogenase could be carried out by co-operation of the non-coupled NADH:ubiquinone oxidoreductase and the bd-type quinoloxidase. Efficiency of this chain seems to be five-fold lower than that of the usual proton-motive chain, which is also present in A.vinelandii and operates at low [O2].
Crystal structure of COX reveals two potential input proton channels denoted as D- and K-channels. We have compared wild type R. sphaeroides COX and mutants with amino acid replacement in these channels: E286, D132 (D-channel) and K362, T359 (K-channel). Effects of the mutations on the spectra, ligand reactivity and peroxidase activity of COX were studied. In the dithionite-reduced E286Q, MCD spectra in the Soret indicate transition of 70-80% of heme a3 from the high to low spin state. MCD of the oxidized E286Q is consistent with hexacoordinated ferric state of heme a3. Accordingly this mutant COX does not react with KCN and H2O2 in the ‘oxidised’ state. Presumably heme a3 in E286Q is trapped at the unprotonated iron(III)-peroxy complex with spectrum of the ferrous-oxy state. CD and MCD data together suggest that replacement E286Q perturbs significantly surrounding symmetry of the both hemes a and heme a3. D132N COX showes decreased reactivity towards H2O2. E286Q COX did not react with H2O2 at all. Reaction of K362M COX with H2O2 at pH above 8 resembles reaction of WT enzyme at acidic pH. Formation of compound P during aerobic CO treatment of the oxidized K362M COX is decreased. Some K362M COX preparations have significant catalase activity. The K-channel mutants although unable to turnover with dioxygen, can utilize H2O2 as electron acceptor with either ferrocyanide or cytochrome c as electron donors. Their peroxidase activity is similar to that of the WT COX and is coupled to D y generation and proton pumping. No peroxidase activity is revealed in the D-channel mutant COX, D132N and E286Q. Reduction by dithionite is severely inhibited in K362M, but not in D132N COX. Mutations in the D-channel arrest COX turnover by inhibiting proton uptake in the peroxidase part of the catalytic cycle while the K-channel is required for the initial reduction of binuclear center in the eu-oxidase phase. Electrometric technique applied earlier in the studies of F->Ox transition has been used to resolve charge translocation coupled to P->F transition. P was generated by aerobic CO bubbling through suspension of COX proteoliposomes in the presence of ferricyanide. As in the case of F->Ox transition, the electric response includes a rapid KCN insensitive electrogenic phase with t of 40-50 m s (reduction of heme a by CuA) and a slower multiphasic cyanide-sensitive part (vectorial proton transfer). The protonic part of the P->F electric response is faster than in the F->Ox transition but much slower than reported for the P->F transition during oxidation of the reduced COX by oxygen. This difference is due to different states of CuB in compounds P formed under these conditions. We also made preliminary experiments on D y generation in the E286D mutant COX. In contrast to the ‘dead’ E286Q mutant, the E286D COX is about 50% active and first single-turnover experiments reveal retardation of the protonic electrogenic phases.
Uncoupling effects of laurate and lauryl sulfate have been studied in isolated rat liver and skeletal muscle mitochondria. It is found that two-fold stimulation of respiration of the oligomycin-treated liver mitochondria, accompanied by a membrane potential decrease, is caused by 2x10-5 M laurate or 1.6x10-4 M lauryl sulfate. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the action of laurate and lauryl sulfate on respiration rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Cations neutralizing negative charges of the membrane surface, such as tetraphenyl phosphonium and cetyltrimethyl ammonium, cause an alkaline shift of these pH dependencies. A small amount of lauryl sulfate, increasing the membrane negative charge, induce the opposite shift. ADP but not GDP induce partial recoupling with both laurate and lauryl sulfate. It is concluded that in liver mitochondria uncoupling by lauryl sulfate, like that by laurate, is mediated by the ATP/ADP and glutamate/aspartate antiporters. The antiporters are assumed to be involved not only in translocation of the uncoupler anions but also in their protonation by extramitochondrial H+ ions.
In skeletal muscle mitochondria uncoupled by laurate, it is found that GDP added before CAtr causes partial recoupling which becomes complete after subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP entails larger recoupling than after GDP and prevents the GDP recoupling. ADP but not CDP substitutes for GDP. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP resistant but sensitive to CAtr, glutamate and serum albumin. It is suggested that a skeletal muscle mitochondria, besides the ATP/ADP and aspartate/glutamate antiporters, a GDP-sensitive mechanism, presumably uncoupling proteins (UCP) 3L and 2, is involved in uncoupling by laurate. Some of these UCPs, in contrast to UCP1, seem to be sensitive to CAtr but, like UCP1, cannot protonate the transported anions such as lauryl sulfate.
During the period covering 1996-1998, different aspects of the mitochondrial permeability transitions (PT), which, from our point of view, represent the functional state of the mitochondrial benzodiazepine receptor, were under study. This study has been split into different projects as follows:
The Project 1 was dealing with the exploring of functional parameters, determining the severity of PT as well as the ways of its proper regulation. Among the factors reasonably capable to regulate the activity of the mitochondrial megachannel, first, the oxygen was chosen. We have demonstrated that in spite of a common view on the obligatory role of oxygen in PT, mediated by reactive oxygen species, this factor seems to be essential but can be bypassed by other stimuli. Nucleic acids were another factor under our decent analysis. According to the hypothesis we put forward, the activity of the mitochondrial benzodiazepine receptor is modulated by nucleic acids. In this project we were able to prove that first, mitochondrial DNA or its fragments are capable of penetrating through the megachannel and second, the structural analogs of nucleic acids as well as viral RNA, both interact with cytochrome c, resulting in its release from mitochondria for possible activation of the apoptotic cascade.
In the Project 2 we were trying to isolate the proteinaceous complex from the mitochondrial contact sites, which is responsible for PT. By the electrophoresis (under native conditions) of the extracts after mitochondria being treated with low Triton X-100, we isolated the proteinaceous supercomplex tentatively containing porin, hexokinase, cyclophilin, adenine nucleotide carrier and creatine kinase. We found the tight functional coupling existing between all components of the complex. The reconstitution of the complex into bilayer membrane resulted in the channel formation, resembling the megachannel observed when PT was activated.
The Project 3 was focusing on the study of PT in a living cell. We
were first who demonstrated that in the primary culture of the rat brain
granule cells glutamate induces the depolarization of the inner mitochondrial
membrane assigned to PT pore opening. The role of Ca and Na ions in the
glutamate toxicity was analyzed. The conclusion has been made that Ca/Na
exchanger when being activated prevents the cell death probably induced
by PT pore opening in neuronal mitochondria.
Effect of a cationic polymer, poly(L-lysine), on the kinetic properties of ionic channels formed by neutral gramicidin A and its negatively charged analogue O-pyromellitylgramicidin 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 gramicidin A channels, but causes a substantial increase in t for O-pyromellitylgramicidin channels. This effect is reversed by addition of polyacrylic acid. The deceleration of the photoinactivation kinetics is ascribed to electrostatic interaction of polylysine with O-pyromellitylgramicidin probably resulting in O-pyromellitylgramicidin clustering. The latter can stabilize the channel state by reducing the rotational and lateral mobility of O-pyromellitylgramicidin monomers and dimers, and thus increase the single channel lifetime.
The concept of strict functional optimization of light-harvesting
photosynthetic antenna structure was put forward several years ago. Using
simulations of excitation energy transfer from antenna to reaction center,
some guiding principles for organization of an optimal antenna model were
identified. Targeted searches for theoretically identified structural optimization
criteria have subsequently allowed one to recognize them in natural photosynthetic
antennae, in particular, in chlorosomal superantennae of green bacteria.
Oligomerization of light-harvesting antenna pigments in vivo was predicted to be biologically expedient being one of efficient strategies for light harvesting. The direct experimental proof for oligomeric organization of chlorosomal pigments in vivo was demonstrated by hole burning in fluorescence and fluorescence excitation spectra of intact cells. Spectral hole-burning studies allowed one to determine the fine structure of electronic spectra of chlorosomal pigments in vivo, that provided us with an exciton level structure criterion for theoretical evaluation of any model for pigment aggregation in the chlorosome. Using the standard approach to exciton-phonon problem in molecular crystals, we discovered that none of the models of pigment aggregation in Cf.aurantiacus chlorosome, proposed for last 20 years, displays the in vivo exciton level structure of the aggregate revealed by hole-burning studies, and, moreover, may serve as prototypes for optimal antennae. Our pump-probe experiments yielded information about Cf.aurantiacus antenna pigments kinetics, absorption difference spectra evolution on femto- and picosecond time scales, and pigment aggregation. We demonstrated that two methods of nonlinear spectroscopy -hole-burning and ultrafast pump-probe spectroscopies, used in combination - allow one to discover key data on both electronic and three-dimensional structure of the antenna aggregates in vivo.
A model of pigment aggregation in the membrane B808-866 antenna of the green bacterium Cf.aurantiacus is proposed. The size of a unit building block of this antenna has been determined.
Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. The two-fold decrease in magnitude of the microsecond photovoltage generation coupled to the M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable with that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of the M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bR ground state and transition of the purple neutral form into the blue acid form. Thus, pK of E204 in the M state is close to pK of D85 in the bacteriorhodopsin ground state (<3) and far below pK of the terminal proton release group (~6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.
Flash-induced voltage response of halorhodopsin at high NaCl concentration
comprises two main kinetic components. First component with t~1 ms does
not exceed 4% of the overall response amplitude and is probably associated
with the formation of L (hR520) intermediate. The second main component
with t~1-2.5 ms which is independent of Cl- concentration can be ascribed
to the transmembrane Cl- translocation during the L intermediate decay.
Photoelectric response in the absence of Cl- has the opposite polarity
and does not exceed 6% of the overall response amplitude at high NaCl concentration.
A pH decrease results in substitution of the Cl--dependent components by
the photoresponse which is similar to that in the absence of Cl-. Thus,
the difference between photoresponses of chloride-binding and chloride-free
halorhodopsin forms resembles that of bacteriorhodopsin purple neutral
and blue acid forms, respectively. The photovoltage data obtained can hardly
be explained within framework of the photocycle scheme suggested by Varo
et al. (Biochemistry 1995, 34, 14490-14499). We suppose that O-type intermediate
belongs to some form of hR incapable of Cl- transport.
The femtosecond spectroscopy experiments at core antenna from Rhodopseudomonas viridis [Novoderezhkin et al. 1998], peripheral antenna from Rhodobacter sphaeroides and dimeric subunite B820 from Rhodospirillum rubrum were carried out. The influence of detergents on linear spectra (absorption and circular dichroism) of LH2 complex from Chromatium minutissimum was investigated. The problem of excitation delocalization in antenna of photosynthetic bacteria was summarized in invited (symposia) report at XIth International Congress on Photosynthesis (Budapest, 1998) and in following full-format paper.
Photovoltage responses accompanying electron transfer on the acceptor side of photosystem I (PS I) were investigated in proteoliposomes containing PS I complexes from cyanobacteria using a direct electrometrical technique. The results obtained suggest that the electrogenesis on the acceptor side of PS I is related to electron transfer between iron-sulfur centers Fx and Fa, as well as between Fa and Fb. The interactions with the water-soluble natural electron acceptors ferredoxin and flavodoxin in control and in the iron-sulfur center Fb-less PS I samples were studied. It is shown that Fb cluster is necessary for the efficient PS I interactions with these electron acceptors. It is concluded that Fb is the terminal iron-sulfur cluster in PS I, and the electron transfer between Fb and the water-soluble acceptors is electrically silent.
1) Recent studies of reaction centers from Rhodobacter sphaeroides (R-26) in which bacteriopheophytins a were replaced by plant pheophytins a, have shown that at low temperature the excited state of primary electron donor P* is converted to the state P+BA- (where BA is a bacteriochlorophyll a monomer in branch A) which has a long lifetime (about 600 ps). This allows the direct measurement of the free energy difference between P* and P+BA- using the temperature dependence of the recombination fluorescence from P+BA-.
The data show that P+BA- is located below P* by 550+-30 mV. Thus the primary conversion of P* leads to the formation of P+BA- which is below P* in energy and is a real intermediate in electron transfer.
2) Femtosecond oscillations of optical density in absorption band and of stimulated emission of primary electron donor P at 860-890 nm and in absorption band of bacterioclorophyll monomer (B) at 800 nm of the reaction centers (RC) Rhodobacter sphaeroides were studied. It was shown that electron transfer from P* to B induced by 15-30 fs excitation is accompanied by coherent nuclear motion with 10-300 cm-1 frequencies. It was found that the P excitation induces both fs oscillation in stimulated emission of P* in the absorption band of B at 800 nm with similar spectra of Fourier transforms.
In the absorption band of B a significant increasing of oscillation amplitudes of the 12 and 30 cm-1 frequencies is observed. It was found that in RC of Rhodobacter sphaeroides mutant YM210W in which a tirosine M210 located near P and B is changed by triptophan, amplitudes of 12 and 30 cm-1 oscillations strongly suppressed in comparison with native RC.
It was found in [Parson et al.] that the motions of nuclear subsystem with 15 and 30 cm-1 frequencies are accompanied by changing a distance between P and B. It is evident that such motions can play a significant role in electron transfer, because in this case the interaction energies of P and B molecules are changed and mutual configuration most optimal for electron transfer is found.
3) The properties of high- and low-spin forms of cytochrome b559 in core complexes of photosystem II of spinatch chloroplast were investigated.
In these complexes the main part of cytochrome is in oxidized state,
and investigation by EPR method becomes possible. It was found that cytochrome
b559 presents in high- as well as in low-spin forms. The spectrum of high-spin
form has a complicated structure with components around g=6 and g=7. The
signal of heme Fe(III) is found as well as the signal of nonheme Fe(III)
with g=8.08. Illumination of the complexes at 50K leads to a significant
decrease of the later signal without changing of heme Fe signal. Decreasing
of heme Fe signal with g=7 takes place under illumination at higher temperatures
(>90K). Under these conditions the component with g=6 is not increased
in contrast to cytochrome b559 in chloroplasts. Study of the core complexes
EPR signal of cytochrome at different pH has shown that signal with g=7
has pK ~ 7, or dissociation constant of Fe(III)-OH-complex equal to 0.1
mmM. Changing of pH leads to completely reversible changes of g=7 component.
The obtained results shows the considerable affinity of heme Fe(III) to
ion OH-. Photochemical conversions of heme Fe(III) liganded by OH- suggest
the possibility of a direct participation of cytochrome b559 in the water
oxidation in photosystem II of green plants.
The three-dimensional structures of E.coli PPase (apo-PPase) and its complexes with Mn2+ in high affinity site and with Mg2+ in high and low affinity sites determined by authors at 1.9-2.2 A resolution have been analyzed in detail. It is found that metal ion binding initiates the shifts of a -carbon atoms and functional groups and rearrangement of non-covalent interaction system of hexameric enzyme molecule. As a result, the apo-PPase with 6 equal subunits turns after Mg2+ binding into the structure with three types of subunits distinguished by their structure and occupancy of the low affinity Mg2+ site. The induced asymmetry reflects the subunit interaction and cooperativity between Mg2+-binding sites. These molecular rearrangements are the structural basis accounting for specific features of the enzyme behavior and suggest one of the pathways for the enzymatic activity regulation of constitutive PPase in vivo.
The further investigations of mutant PPases with Asp 42 substituted have been carried out. This residue is proposed to be situated nearby the fourth Mg2+ binding site. The kinetic parameters of PPi hydrolysis by Asp-42Glu and Asp-42Ala mutant PPases have been determined. Km values of all variants with Asp-42 mutated (by Asn, Glu and Ala) are essential increased in comparison with native enzyme. All of them lose the ability to inhibit by Mg2+ at its high concentration. These results support the proposal that Asp-42 is essential for the substrate and Mg2+-inhibitor binding site formation. Asp-42 mutated PPases interaction with Ca2+ has been studied by kinetic and equilibrium dialysis methods. It has been shown that all variants except Asp-42Ala retain two Ca2+ binding sites in substrate absence; one of them can be substituted by Mg2+. Asp-42Ala PPase has an additional Ca2+ binding site. It has been proposed that Ca2+ firstly displace Mg2+ from substrate bound and then from magnesium binding site on enzyme.
The further realization of Ca2+ role demanded the knowledge of three-dimensional structures of PPase complexes with Ca2+ and substrate. X-ray analysis at 1.2 A resolution of Ca2+- and CaPPi- PPase complexes has been carried out.
The formation of enzyme-substrate complex stabilized by F- has been investigated for the mutant E.coli PPase with Asp-67Asn substitution. Mg2+-supposed PPi hydrolysis by the Asp-67Asn PPase at saturating PPi concentrations in the presence of F- is associated with gradual decrease in the initial rate of PPi hydrolysis. The PPase inhibition by F- occurs in two steps. The immediately added inhibitor binds to the enzyme-substrate complex; this process is followed by the slow conformational changes in the enzyme-substrate compound resulting in activity loss. This compound is partially active and can be isolated by the removal of the inhibitor by gel-filtration and used for X-ray analysis.
For bakers' yeast PPase, its monomeric form obtained by mutating Trp279 in the subunit contact zone was characterized functionally. The monomeric form exhibits by about 20-fold reduced affinity of free enzyme to magnesium ions and the rate constant for substrate binding. In this respect, yeast PPase resembles E. coli PPase, but the effects of enzyme dissociation are much smaller. Lys193 substitution has little effect on PPase interaction with phosphate in the presence of Mn2+. The dissociation constant for enzyme-phosphate complex under optimal conditions approaches 10-8 M. Asp117, Asp120 and Asp152 substitutions in PPase active site change its inhibition by fluoride. These data suggest that fluoride displaces Wat1 water molecule, which is linked directly or through metal ions to all these three residues in enzyme structure.
Two new electrophoretically homogeneous preparations of cationic inhibitors BWI-1c and BWI-3c were obtained from buckwheat seeds. The molecular masses of the inhibitors were equal to 6 kD according to PAGE-analysis. It was established that BWI-1c contains an Arg residue at P1 position of the reactive site and BWI-4c - a lysine residue. Both cationic inhibitors were highly pH- and thermostable. Both inhibitors effectively suppressed trypsin, while BWI-3c inhibited bacterial subtilisin and subtilisin-like proteinases as well. Complete amino acid sequence of BWI-4a protease inhibitor from buckwheat seeds was established by mass-spectrometry and automatic Edman degradation. The protein consists of 67 amino acid residues, has a residue of pyroglutamic acid at the N-terminus and belongs to the family of proteinase inhibitor I from potatoes. The N-terminal sequence of I9 amino acid residues of cationic inhibitor BWI-1c has been established by autimatic Edman degradation. Inhibition constants of bovine trypsin and chymotrypsin by protease inhibitors from buckwheat seeds BWI-1a, BWI-1c, BWI-2c and BWI-4c and association and dissociation constants of BWI-1a inhibitor with bovine trypsin were established. A highly purified (311-fold) preparation of a serine proteinase secreted by filamentous fungi Trichoderma harzianum has been obtained and some of its properties characterized.
The main directions of the work in 1998 were synthesis and characterization of the reagents for affinity modification of EcoRII restriction endonuclease (R.EcoRII) and EcoRII DNA methyltransferase (M.EcoRII); obtaining of DNA-protein conjugates and creation of system for site-directed mutagenesis of R.EcoRII. 14-mer DNA duplexes, EcoRII enzymes substrate analogs, have been synthesized, which contain photoactivated aryl(trifluoromethyl)diazirine(ATFMD)group in heterocyclic base in the center of the recognition site or reactive dialdehyde group in the sugar-phosphate backbone of the flanking nucleotide sequence. The DNA duplexes stability have been studied in UV melting experiments. Binding of both types of reagents to R.EcoRII as well as their substrate properties have been examined. It was shown that incorporating modified groups does not preclude from forming enzyme-substrate complexes. However, cleavage or methylation of modified DNA duplexes are strongly reduced. Thus, R.EcoRII cleaves ATFMD-containing DNA duplex only in the presence of canonical substrate. Cross-linking of base -modified 14-mer substrate to EcoRII endonuclease or methylase is effected by irradiation of the enzyme-substrate complexes at 366 nm. Effective cross-linking of the DNA duplexes with periodate - oxidized galactose residues to M.EcoRII and R.EcoRII has been performed in a variety of conditions. The methods of purification of conjugates of the enzymes with both reagents have been developed. A putative R.EcoRII active site motif has been chosen for site-directed mutagenesis. The system for effective purification and analysis of R.EcoRII mutants as hexahistidine derivatives has been obtained.
The kinetic model of the interaction between apotransketolase and coenzyme has been worked out. The model takes into consideration the presence of two active sites and the fact that the process includes two steps. The reason for the non-equivalence of the transketolase (TK) active sites in their affinity to the coenzyme (thiamine pyrophosphate, TPP) has been clarified. A simple and complicated kinetic model of the TK reaction has been worked out. The kinetic parameters of the individual steps have been defined. The competition between donor and acceptor substrate in catalysis has been studied. It has been discovered that the transketolase reaction can proceed in the absence of acceptor substrate (in the presence of donor substrate only). The rate constant of the limiting step of this reaction has been defined.
It was shown that the HO-group at the C-2-atom of the acceptor substrate
takes the immediate part in its interaction with TK by the formation of
a bond with Asp477. The His103 residue of TK participates in the donor
substrate binding by interacting with its C-1 hydroxyl. Besides, His103
influences the intermediate of the TK reaction (glycolaldehyde residue):
it is either (when His103 is intact) transferred to the acceptor substrate
or is released free into the medium. The fact of Glu418 participation in
the TK catalysis has been experimentally proven. It has also been found
that hydroxyphenyl pyruvate, a natural analogue of one of the TK substrates,
can regulate both the TK activity and the tyrosine biosynthesis.
The total goal of the project was to clarify the post-translational mechanisms controlling stoichiometry and intracellular contents of mitochondrial cholesterol hydroxylase system (cytochrome P450scc, adrenodoxin, and adrenodoxin reductase) and those of some monooxygenase systems of the endoplasmic reticulum (CYP2D6 and CYP 3A4). To this end we constructed yeast strains capable of effective synthesizing individual components of these systems and some their hybride forms under the control of artificial regulated promoters. Intracellular location of the foreign proteins, their proteolytic processing, folding, and ability of taking catalytically active forms were under study. Our main concern was with the interaction of the foreign proteins with chaperones and proteolytic systems of recipient mitochondria. Besides, we have shown that the yeast strains producing human cytochrome P450s can be employed as the tools in studies of the mechanisms of their induction by various substances. Following the work done, positive and negative aspects of the heterologous models (yeast cells producing mammalian proteins) used for the solution of the aforesaid problem were revealed and the ways of their improvement were projected.
The use of proteases as catalysts of peptide synthesis is a promising direction of modern peptide chemistry. In a kinetically controlled process of peptide bond formation from peptide esters and aminocomponents one of the major obstacles is a secondary hydrolysis of the product. Peptide synthesis in organic solvents with low water content helps to solve this problem.
The influence of reaction mixture composition and enzyme state on enzymatic peptide synthesis was investigated. The solubility of subtilisin 72 in some organic solutions was studied. Subtilisin 72 was shown to be able to catalyze the peptide bond formation when suspended in acetonitrile. The dependence of product yield on time and enzyme concentration were studied. It was shown that subtilisin 72 in a complex with sodium dodecylsulfate (SDS) is soluble in ethanol and propanol. For the first time SDS-subtilisin was applied for the synthesis of a series tri-, tetra- and hexapeptides and the oligomerization reaction of peptides. The using of SDS-subtilisin for enzymatic peptide coupling on solid support was shown. The synthesis of peptidyl aldehydes via enzymatic acylation of semicarbozons of aminoaldehydes was developed. The reactions were performed in organic solvents using as a catalyst subtilisin 72 distributed on the surface of macroporous silica. The inhibition effect of peptidyl aldehydes on hydrolysis of specific chromogenic substrates by chymotrypsin and subtilisin was studied. The inhibition constants for chymotrypsin and subtilisin were determined.
A hypothesis has been proposed and experimentally proven that the efficiency of conversion of 1,3-diphosphoglycerate via glycolytic or 2,3-diphosphoglycerate path in erythrocytes is determined by the formation of bienzyme complexes between the proteins participating in these reactions. It was shown that complexes between D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (in its dimeric form) and 3-phosphoglycerate kinase or 2,3-diphosphoglycerate mutase exist in human erythrocytes. The complexes can be extracted from erythrocyte hemolysates with the use of immobilised monoclonal antibodies capable of interacting with dimeric GAPDH forms, but not binding the tetrameric forms of this enzyme.The influence of the antibodies on the GAPDH activity, as well as peculiarities of their interaction with different oligomeric enzyme forms was investigated.
It was found that increasing pH of the blood plasma resulted in an increase of the concentration of the complexes GAPDH · 3-phosphoglycerate kinase in erythrocytes whereas upon lowering pH, increased formation of complexes between GAPDH and the mutase occurs. Removing the bienzyme complexes from erythrocyte hemolysates with the use of monoclonal antibodies hinders the accumulation of 2,3-diphosphoglycerate at pH 6.5 and of 3-phosphpoglycerate at pH 8. The results obtained indicate that the bienzyme complexes detected in our study participate in the regulation of 2,3-diphosphoglycerate formation and provide evidence for marked pH-dependence of this process.
Uncoupling of oxidative and phosphorylating steps of glycolysis in
the presence of micromolar concentrations of hydrogen peroxide was demonstrated,
and the role of GAPDH sulfhydryl groups in this process was established.
It was shown that recoverin preferably inhibits unbleached rhodopsin phosphorylation than it does that of bleached rhodopsin. We hypothesized that this preference might be used by a retinal rod cell to prevent rhodopsin kinase from participating in a side reaction of the non-stimulated receptor phosphorylation and to reserve the kinase for fulfiling its essential physiological function of quenching (desensitization) the stimulated receptor activity.
Several EF-hand recoverin mutants have been obtained and their functional characteristics have been determined. It was found that the mutants -EF2, -EF3 and -EF2,3 with the "spoiled" 2nd, 3rd or (2nd + 3rd) EF-hand structures correspondingly did not act upon the kinase activity in the m M range of Ca2+ concentrations. Mutations of the 4th EF hand, which "repaired" its Ca2+-binding activity, gave the mutant +EF4 with three "working" Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild type recoverin (wt-recoverin), containing two working Ca2+-binding structures.
Wt-recoverin and its EF hand mutants have been studied by intrinsic
fluorescence and circular dichroism spectroscopy. We demonstrated that
the binding of Ca2+ to the 2nd and 3rd
working Ca2+-binding sites of wt-recoverin makes the environment
of its tryptophan residues more flexible and shifted its thermal denaturation
transition toward higher temperatures. The amino acid substitutions in
the 2nd and 3rd EF hands decreased their Ca2+
affinities to different extent: the high Ca2+ affinity of the
mutant -EF3 was totally abolished, in comparison with wt-recoverin, whereas
-EF2 demonstrated only a moderate decrease in the affinity. We suggested
that the binding of Ca2+ to recoverin is sequential and that
the 3rd EF hand being filled the first. The recoverin
mutant +EF4 with three working Ca2+-binding sites demonstrated,
in comparison with wt-recoverin, increased Ca2+ capacity and
affinity, disturbed tertiary structure and decreased thermostability of
In 1991 we described a novel Ca2+-binding protein, recoverin which was demonstrated to be highly specific for retina. Recently it was found that early stages of a severe and widespread human tumor, small-cell lung carcinoma (SCLC) could be accompanied by appearance of antibodies against recoverin in the patients blood and by a loss of their eyesight (syndrome named cancer-associated retinopathy). In 1998 we started a research of the recoverin expression in SCLC cultures. In addition screening of patient's blood with ophtalmological, pulmonological and oncological diseases are being carried out to estimate recoverin as a potential marker of SCLC.
Studies of the functional role of the calcium ion-binding site recently discovered in the enzyme structure by enzyme crystallography group at the York University were initiated. Kinetic studies have demonstrated that binding of the metal ion does not influence seriously stereospecificity of penicillin acylase catalysis, i.e. this ion does not play a role of the positive charge in the penicillin acylase active center responsible for chiral discrimination of the substrate. Calcium ion binding site was demonstrated to be responsible for stability of penicillin acylase; moreover this site can be occupied by different metal ions and observed stabilization effect was dependent on the nature of the metal ion. Investigation of the role of the metal ion-binding site in penicillin acylase catalysis is in progress.
Important step in the development of the new method of kinetic investigation of hardly detectable ("invisible") enzymatic reactions, which can not be studied using traditional approaches, was carried through. The method is based on simultaneous run of two competing reactions catalyzed by the same enzyme: "visible" one (for example, conversion of colorimetric substrate) and "invisible" reaction (conversion of unknown substrate, when reaction itself is not followed by detectable spectrophotometric changes). In the case when both reactions are performed in the same reaction vessel colorimetric product accumulation curve does contain information related to the «invisible» reaction. Kinetics of the conversion of new colorimetric substrates by penicillin acylases from Escherichia coli and Alcaligenes faecalis was studied and the most specific "visible" substrates for application of the new method for kinetic studies of penicillin acylase substrate specificity were chosen. High sensitivity of both penicillin acylases to the structure of the colorimetric substrate leaving group was observed indicating the importance of electrostatic interactions at the corresponding subsites of the enzymes.
A study of mRNA path through the ribosome using photoaffinity cross-linking approach was performed. Two new photoaffinity analogues (6-thioguanosine and diazirine derivative of 5-methyleneaminouridine) were applied. They were incorporated randomly into mRNA chain in the course of T7 transcription. After the isolation mRNA was subjected to further modification in the case of diazirine derivatives. The complexes with ribosomes, modified mRNA and tRNAfMet were formed and subjected to mild UV irradiation. After isolation and purification of the complexes positions of the cross-links were identified. Several new cross-links have been found. 6-thioG at positions +8,+9 of mRNA forms cross-link with nucleotide 1196 in the helix 34 of 16S rRNA and at position +12 - with residue 530 in helix 18. The application of the diazirine derivative of uridine residue allowed us to find new very important contact of the mRNA residue +2, a part of codon-anticodon duplex, with G926, a conservative nucleotide bulged out helix 28 which connects the "head" and the "body" of small ribosomal subunit. The data obtained together with literature data provided a basis for molecular modelling of the mutual arrangement of mRNA and helices of 16S rRNA as well as ribosomal proteins. This model allows not only to refine the positioning of the 16S rRNA elements at the decoding centre but also to speculate about possible conformational changes in 16S rRNA structure at the decoding region which could occur during the translocation catalysed by the elongation factor G.
To determine the contacts of selected nucleotide residues of 16S
or 23S rRNA with ribosomal components we have developed procedure for insertion
of the photolabel to a selected site of large ribosomal RNA. rRNA was cleaved
at a single selected internucleotide bound and the photolabel, 4-thiouridine
diphospate, was ligated to the 3’-end of a fragment at the cleavage site.
Such fragmented and modified rRNA was reconstituted with ribosomal proteins
into active ribosomal subunits which were subjected to mild UV irradiation.
Resulted cross-links were analyzed using the combination of RNase H and
primer extension analysis. To test the validity of the approach on one
hand and to understand the structure of the interesting rRNA region on
the other we have started with the cross-linking of the photolabel attached
to position 1141 in helix 39 of 16S rRNA. We have found two internal cross-links
to U1295 and G1272, both nucleotide residues belong to helix 41 of 16S
rRNA. The proximity of helices 39 and 41 were established earlier. Thus
we have proved that the method developed provides a valid information about
the contacts in 16S rRNA inside the ribosome. On the other hand, two point
contacts in helix 41 with one residue in helix 39 allowed us to model folding
and mutual orientation of these helices in the 30S subunit. The method
described above was applied for investigation of 23S rRNA structure in
the ribosome. It was shown that thioU residues attached to nucleotides
at position 384 was cross-linked to nucleotide residue 420, at position
867 – to 909 and 917, at position 1045 – to 993, and at position 1117 –
to 1037. The data obtained provide us with unique information about mutual
arrangement of 23S rRNA structural elements in the ribosome. The procedure
developed is very useful for obtaining the data about structural features
of large RNA molecules, especially outside of low molecular weight ligand
binding regions as well as for monitoring conformational changes in rRNA
structure during ribosome functioning.
Topography of 5S rRNA inside the ribosome has been investigated by photoaffinity cross-linking approach. 5-methyleneamino-uridine or- 2’-amino-uridine has been randomly incorporated in 5S rRNA during T7-transcription. Such 5S rRNA was subjected to further modification to incorporate diazirine photoaffinity group. After the reconstitution of the ribosomes with modified 5S rRNA they were subjected to mild UV irradiation to generate chemical cross-links. The cross-links between 5S rRNA and 23S rRNA were analysed with a help of technique developed in our laboratory. We have found that U89 of 5S rRNA forms several cross-links within domain II of 23S rRNA. These data are very important for understanding the spatial arrangement of 23S rRNA molecule around 5S rRNA inside 50S ribosomal subunit.
Site-directed mutagenesis was applied for the investigation of the functional role of A960 23S rRNA, which forms the cross-link with U89 5S rRNA. Substitution of A960 for C or U significantly reduces the cell growth rate. Application of chemical and enzymatic probing allowed us to show that these mutations in domain II affect significantly the structure of peptidyltransferase ring in domain V of 23S rRNA.
In order to study the contacts of sugar-phosphate backbone with ribosomal components thiophosphate groups were randomly incorporated into 5S rRNA molecule in the course of T7 transcription. After the formation of 5S rRNA-protein complex and reconstitution of 50S subunit or 70S ribosome they were subjected to iodine treatment to cleave RNA chain at the position of thiophosphate group. If the phosphate group is involved in tight interactions with protein or RNA, it is protected from the iodine cleavage. As a result we have obtained new data about fine structure of 5S rRNA inside the ribosome, its contacts with specific ribosomal proteins and reveal the possible intersubunit contact.
The combination of different approaches allowed us to develop the
structural model of 5S rRNA in the ribosome and to get new information
about functional role of 5S rRNA and individual rRNA nucleotide residues
in ribosome functioning.
The strategy was developed to synthesize the new types of preactive hairpin-shaped DNAs for crosslinking with proteins recognizing specific nucleic acid sequences. Intramolecular DNA duplexes were chosen as the decoys targeting human transcription factor NF-kB because of their increased thermal stability and nuclease resistance compared to regular double-stranded oligonucleotides. Using chemical ligation method, the internucleotide sulfur-containing reactive linkages were introduced instead of native phosphodiester ones into DNA sugar-phosphate backbone. Since the variety of modified residues facing the nick determined the type of newly formed internucleotide linkage, oligonucleotides carrying 3’(5’)-terminal phosphate, thiophosphate, S-alkylthiophosphate or 2-pyridyldisulfide groups were prepared by automatic synthesis and postsynthetic procedures. These oligonucleotides was shown by UV-spectroscopy and chemical assays to form five families of nicked regular and hairpin-shaped DNA duplexes differing in sequence, nick position and position of AAA base loop connecting the ends of complementary strands. Effective chemical ligation approaches were developed which allow to introduce diphosphoryldisulfide groups and sulfur-containing pyrophosphate analogs at predetermined position of DNA sugar phosphate backbone. Spontaneous and template-controlled exchange of oligonucleotide fragments joined by disulfide bond was studied. Chemically active DNA duplexes with trisubstituted thiopyrophosphate linkages in two different configurations were prepared. The efficiency of water-soluble carbodiimide-induced ligation was shown to depend on position (3’ or 5’) of S-alkylthiophosphate group in the nick. It was found that modified DNA duplexes could react with propylenediamine or dithiothreitol molecules imitating lysine and cysteine residues. These properties make prepared chemically active hairpin DNAs quite useful reagents for affinity modification (inhibition) of regulatory proteins containing lysine and cysteine residues in the binding sites.
Sequences of ITS 2-4 region (23S-tRNA arg) of chloroplast rRNA operon from several dozen species of seed plants were determined. The main conclusions inferred from data on different types of ITS's and from different estimates of genetic distances are in a good agreement. Results of computer analysis provide strong bootstrap support for monophyly of both spermatophytes and angiosperms. A weaker support for monophyly of gymnosperms studied and for a basal position of woody Magnoliales + Nympheales among angiosperms on phylogenetic tree was observed. There is no sister group relationship between any gymnosperm taxon including Gnetales and angiosperms on the basis of analysis ITS 2,3,4 regions as well as rbcL gene sequences (literature data). Divergence of main groups of extant gymnosperms occured after branching off angiosperm line.
Analysis of ITS 2-3 region of ferns supports their closer relatedness to seed plants than to bryophytes. ITS 2,3 and 4 sequences of cpDNA of several dozen of bryophyte species and of Chara australis (outgroup) were determined. An interspecies length heterogeneity of regions under investigation was detected. Bryophytes form two clades (Bryophyta and Jungermanniophyta), and the first of them includes all the vascular plants. Hence, bryophyte system needs to be radically revised. Basing on ITS 2,3 and 4 data the first comlete phylogenetic tree of land plants was constructed.
4.5S rRNA gene was found in cpDNA of Chara australis. It follows
from this fact that land plants and chara-like algae had a common ancestor.
ITS1, ITS2 and 5.8S rDNA from fungal nuclear genome of 20 Umbilicariaceae
lichen species were sequenced. Phylogenetic tree was inferred from these
data. Introns in 18S rDNA of Umbilicariaceae species were localized and
The relationships among nematodes were studied by 18S rRNA gene sequencing. On the basis of phylogenetic trees and cladistic analysis of the secondary structure of helix 49, some orders of traditional subclass (subphylum) Adenophorea should be ascribed to the Secernentea. The Chromadorida and Desmodorida should be grouped with nematodes of the complex consisting of the Monhysterida, Plectida and Secernentea. This taxon may be named Chromadoria, since chromadorids are most closely related to the common ancestor of these groups. Hence, the class Adenophorea in a traditional sense is paraphyletic group and should be revised.
Analysis of the secondary structure of 18S rRNA molecules in various
invertebrate taxa revealed some new traits in hairpins 17, 42-44 and 49.
Such traits of hairpins 42-44 convincingly support the closest relatedness
of Cnidaria and Placozoa with bilateral animals. Some of the peculiarities
of hairpin 49 allow resolving the filiation within Nematoda. Hairpin 17
possesses traits some of which are characteristic of all the nematodes,
whereas the others are characteristic exclusively to the order Rhabditida.
The loss of a nucleotide pair in the highly conservative region of the
hairpin 17 distinguishes 18S rRNA of the Strongylida and some species of
the Rhabditida from other nematodes and, moreover, from that of all other
organisms. Therefore, it is possible to regard the Strongylida and a part
of the Rhabditida including Caenorhabditis elegans as a new monophyletic
Genomes of 21 Insectivora species, 4 Artiodactyla species, 4 Primata species (including Homo sapience) were compared by modified restrction endonuclease analysis method (taxonoprint). Digestion of genome DNA with short-cutting endonucleases with the following isotopic end-labeling of the restriction fragments and their separation in non-denaturating polyacrylamide gel was used. This approach enables us to visualise patterns of DNA repeat bands 30-500 bp long. These band patterns showed species specifity rather than individual or population specifity. Phylogenetic trees were constructed on the basis of the number and distribution of bands using the method of maximum parsimony and bootstrap analysis. Erinaceidae species and genera appear to be more distant from each other than species and genera of Soricidae and Talpidae. Intergenetic level of divergence in the two latter families is nearly the same. The degree of divergence of repeated DNA fragments in Insectivora correlates with the amount of geterochromatin in chromosomes of its representatives and with geological age of species.
The related species and intraspecies forms of bisexual eucaryotic organisms differ not so much by the sets of unique sequences but mostly by repeated sequences distribution in genome. Therefore, solving of this problem by the restriction endonuclease analysis modification is useful Now the repetitive genome DNA of the Atlantic salmon fishes and Pacific salmons and trout (16 species and forms) was studied by the method of "taxonoprint". The results for each restriction endonuclease were represented as the matrix, consisting of zeros and unities were the rows corresponded to the investigated species and columns - to zones with the same electrophoretic mobility. These zones were considered to be the binary cladistic characters. The computer programs PHYLYP 3.5 and VOSTORG were used for the generation of unrooted trees and dendrograms. Electrophoretic patterns of DNA are clearly parted into the four groups which have the status of the genus:
1. Salmo salar (Atlantic salmon);
2. The trutta group ;
3. The american and kamchatka forms of Salmo mykiss and Salmo clarki;
4. All 6 species of the genus Oncorhynchus.
Therefore, the including of Pacific trout into the genus Oncorhynchus
is not confirmed by the our molecular genetic data.
The ribosomal biogenesis is a key process of rapidly growing cell. A biogenesis regulation in E.coli cell is governed by two types of the very specific RNA-protein interactions: S7 – 16S rRNA and S7 – str mRNA.
The conditions for overexpression of the recombinant 6His tag protein S7 of Thermus thermophilus in E.coli and stable maintenance of the overexpressing M15 strain have been elaborated.
The thermophilic protein is able to form a heterologous complex with regulatory S12-S7 intercistronic region of str mRNA of E.coli. The conditions of the complex formations have been optimized, and they do not coincide with the conditions of integral ribosomal small subunit reconstitution.
The PCR deletion analysis has revealed that the minimal mRNA fragment able to bind S7 has a size in a range of 63-103 nucleotides, and is larger then proposed S7 binding site footprinted by Nomura. A dot-matrix secondary structure analysis of RNA has yielded a hypothesis on a mode of "feedback" regulation of the coupled translation of two neighboring cistrons of the str operon by the protein S7.
For RNA aptamer SELEX, BLMA amylase has been chosen as a model protein, which does not have natural RNA binding properties. After several rounds of SELEX, an enriched RNA fraction has been selected able to bind the protein to a small extend. Some late rounds of SELEX have been performed under more stringent conditions and an enrichment of RNA aptamer fraction has been achieved.
Tetracycline is an antibiotic which target is a ribosome. A previous literature data pointed S7 RNP domain of the small ribosomal subunit as one of a structural element being modified after direct UV irradiation of the ribosomal complex with tetracycline. Though a number of other sites, including large subunit, have been modified as well.
Gaining to have more precise data, the tetracycline repressor protein TetRD , with known three-dimensional structure of complex with tetracycline, has been selected as a model protein to explore a power of tetracycline as a photoaffinity reagent. The complex of radioactive 7-[3H]-tetracycline with the repressor protein has been obtained. Irradiation of the complex with 250W arc mercury lamp, emitting 366 nm light, has yielded a covalent attachment of the tetracycline to the amino acid residues at the tetracycline binding site of the protein, according to the X-ray data. For the first time, a direct correlation of both crystal and solution data have been revealed. Therefore the tetracycline can be used for the photoinducible affinity modification to study active sites of other tetracycline targets.
The method of induction of multiple DNA-protein and protein-protein
cross-links in chromatin of isolated nuclei has been developed. The cross-linking
is brought about by mutual effect of fluorochrome ethidium bromide (EB)
and irradiation with visible light (350-550 nm). Electron microscopic analysis
of nuclei isolated in solutions with various concentrations of divalent
cations, stained with EB and irradiated with visible light, has demonstrated
that the method leads to the selective stabilization of macromolecular
complexes of chromatin against various treatments causing decompactization
of native chromatin. On the other hand, the same treatment does not stabilize
elementary chromatin fibers (about 30 nm thick), and the transition to
the nucleosomal fiber occurs after extraction of histone H1 with 0.6 M
NaCl. Electrophoretic analysis of proteins from control and irradiated
nuclei shows the basic role of non-histone proteins in the stabilization.
The procedure of photo-stabilization was used to detect additional nuclear
matrix attachment sites, that cannot be revealed by convemtional methods.
These sites was shown to participate in compactization of large loop domains
of DNA in native chromatin.
The cDNA clone corresponding to Xenopus maternal mRNA, Eg7 was isolated. It encodes a protein which expression is strongly increased during oocyte maturation. Immunolocalization of pEg7 indicates that the protein concentrates on chromosomes during mitosis in cultured cells and on chromosomes formed in cytostatic factor-arrested cell-free extracts. Immunodepletion of the extracts with anti-pEg7 antiodies totally prevents in vitro chromosome condensation. Biochemical analysis shows that pEg7 assocoates with Xenopus SMC-proteins, members of 13S condensin.
A novel class of DNA and RNA duplexes including trisubstituted pyrophosphate internucleotide groups in sugar-phosphate backbone was employed in cross-linking to protein. Substrates including this group bound covalently with high efficiency to nucleophilic aminoacids (Lys, His) of transcription factors HNF-1 and NF-kB and to HIV-1 Tat protein. The covalent binding of the protein to DNA or RNA duplexes with trisubstituted pyrophosphate internucleotide linkage proceeded at physiological conditions without additional activation. Thus, such compounds may be used in vivo experiments and may find application in several branches of sense biotechnology.
In order to extend research into this area, we have designed certain cross-linked DNA duplexes including DNA dumbbells. We proposed a new, simple and highly effective method for synthesizing covalently cross-linked DNA duplexes based on the use of oligodeoxyribonucleotides modified at defined sites: one containing 2’-amino-2’-deoxyuridine, the other having a non-nucleoside insertion containing a carboxyl group. We demonstrated that these cross-linked DNA duplexes are strongly resistant to the action of exonucleases and interact with a dimer of the NF-kB p50 subunit. In particular case, cross-links can promote the process of protein binding. These results suggest that cross-linked DNA duplexes are effective inhibitors of NF-kB and may be decoys for transcription factors.
The rational design of initial DNA dumbbells for efficient chemical synthesis of closed ones was developed. The possibility of using DNA dumbbells for probing local unwinding of double-stranded DNA during the functioning of type II restriction-modification enzymes was shown. The DNA dumbbells containing the active acylphosphate internucleotide groups in definite positions of sugar-phosphate backbone were obtained at the first time.
Mapping the binding site of the large subunit (eIF-4G) of cap-binding
factor eIF-4F within the IRES-element of encephalomyocarditis virus RNA
(EMCV) has been performed. As we have earlier shown, this subunit plays
a key role in binding the IRES of EMCV RNA with 40S ribosomal subunits.
Using chemical and enzymatic probing, it has been established that eIF-4G,
both individually and as a part of the whole factor eIF-4F, highly specifically
binds the oligoA-loop confined between three short stems of J-K-domain
of the EMCV IRES. This domain is a proximal one to the initiation codon
of EMCV RNA. In addition, we have studied the molecular mechanism of functioning
of two other IRESs, hepatite C and Classical Swine Fever Virus, that are
not related to picornaviruses. These IRESs are structurally quite different
from picornaviral IRESs. It turns out that to form initiation complexes
these IRESs do not require at all factors 4A, 4B or 4F. Their binding with
the 40S ribosomal subunit does not involve ATP hydrolysis and, like in
prokaryotes, requires just two factors, eIF-2 and eIF-3 as well as the
initiation tRNA. The main role in primary binding of these IRES-elements
with the 40S ribosomal subunit belongs to the 40S subunit itself since
even in the absence of initiation factors and methionyl-tRNA it forms with
the IRESs fairly stable complexes.
Nuclear DNA regions ITS1 - 5,8S rDNA - ITS2 from 40 additional Umbelliferae species were sequenced. Data were combined with those obtained earlier and used for phylogenetic tree reconstruction. The combined set and resulting trees consist of 174 species from Umbelliferae and related families. Comparisons between molecular data based evolutionary tree and the phylogenies inferred from the distribution of several phytochemical and morphological characters show that systems of Umbelliferae classification based on the latter characters are unsatisfactory, especially if the tribes relationships and their size are concerned.
Cytosine DNA-methyltransferase was isolated from wheat seedlings
and purified in the presence of different protease inhibitors. It has molecular
mass and specific activity equal to about 85 kDa and 250 units/mg protein,
respectively. It was shown that wheat DNA methyltransferase is capable
to methylate both hemimethylated and unmethylated double-stranded DNA in
CG and CNG sites. The enzyme loses rapidly its methyltransferase activity
in the absence of substrates. It was found that rapid enzyme inactivation
is due to disappearance of essential enzyme SH-groups but not to proteolytic
cleavage. It was shown that CCGG methylation pattern of coding part of
cytosine DNA-methyltransferase gene is similar in all organs studied (root,
stem, leaf, flower, cod) both in young and aged Arabidopsis plants.
The apoptosis was discovered in wheat plants. The specific apoptotic internucleosomal
nDNA fragmentation (180± 10 bp)n, n³ 1, and an intensive
incorporation of labeled precursors into «heavy» (r = 1.716
g/cm3) DNA fraction were detected in aging coleoptile and initial
leaf of wheat seedling. Unlike nuclear DNA, H-DNA does not contain 5-methylcytosine
but seems to contain N6-methyladenine and is formed
in mitochondria as a result of unusually active synthesis in aging cells.
H-mtDNA formation is the specific programmed and synchronously realized
event in the aging cells of the whole plant organ. The appearance and accumulation
of H-mtDNA in plants can be the indicators of aging. It was shown that
BHT (ionol, 50 mg/l) strongly inhibits growth of etiolated wheat seedlings
but it slows down the coleoptile senescence and induces synthesis of carotenoids
that accumulate mainly in roots. Thus, antioxidants, and BHT, in particular,
effectively regulate the in vitro and in vivo plant growth
and development. Similarly to other known plant growth regulators, antioxidants
may have different and even opposite effects depending on antioxidant concentration
in a medium and cultivation conditions such as temperature.
An analysis of structure-function relationship in a nuclear
protein prothymosin alpha has been performed. Functionally important determinants
in prothymosin were defined, including the nuclear localization signal
of the protein responsible for nuclear accumulation of prothymosin. However,
an unexpected mechanism preventing nuclear uptake of prothymosin was also
disclosed. This novel pathway withdraws a long-standing contradiction between
the ability of prothymosin to be imported into the nucleus and its participation
in intracellular signaling of hemopoietic cells. A specific yeast cell
division arrest caused by human prothymosin was studied in detail. A point
mutation in prothymosin abrogating its inhibitory phenotype was identified.
Several prothymosin-interacting proteins, including hnRNPA1 and Ik Ba ,
were identified with the help of the yeast two-hybrid system. Besides,
prothymosin was demonstrated to link tRNAs covalently; several tRNA attachment
sites were located in the prothymosin molecule. Zinc-binding properties
of prothymosin were observed and studied; both tRNA attachment and protein
binding to prothymosin are stimulated in the presence of zinc ions.
The procedures for isolation and further purification of telomerase complex from Saccharomyces cerevisiae were developed. To test telomerase activity we used the property of telomerase to elongate specific DNA-primer. Partially purified telomerase complexes were applied for the investigation of additional enzymatic activities and protein content of telomerase complexes at different purification stages.
Different techniques were tested to purify telomerase complex. We have found that the combination of two techniques such as glycerol gradient centrifugation and DEAE-sepharose column purification are the most effective to keep telomerase active in primer elongation reaction. After the first stage glycerol gradient fractions were collected and used either for investigation of protein content by photoaffinity cross-linking and enzymatic studies or for further purification on DEAE-agarose column. Telomerase fractions were eluted from the column using gradient of salt concentration in the elution buffers. Telomerase activity was tested in each fraction and fractions with active telomerase were then also used for cross-linking experiment or functional studies.
Application of photoaffinity cross-linking allowed us to detect that telomerase protein content changes significantly with the increase of salt concentration. The highest number of proteins cross-linked to growing primer were detected in the low salt fractions, their number were decreased with the increase of salt concentration.
We have shown that the complex with the highest number of cross-linked protein can make several rounds of primer elongation. High salt telomerase fractions lacking some protein subunits can elongate the primer only once. We have also shown that telomerase complex possesses nuclease activity, which helps telomerase to elongate non-specific primers.
Thus, we have shown that yeast telomerase is a complex multisubunit ribonucleoprotein which have different enzymatic activities.
1. The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is studied with an emphasis on tobamoviruses. The major attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple gene block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for the 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; (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.
2. 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, being infectious to intact plants. It has been suggested that P30-TMV RNA complexes could be converted into the translatable and replicatable form in planta in the course of passage through plasmodesmata. In the present work the role of TMV MP phosphorylation was investigated in terms of its capacity to modulate the translation-repressing ability of the MP. It was found that phosphorylation of TMV 30k-Da MP either before or after RNP complexes formation caused a convertion of nontranslatable MP-RNA complexes into a form that was translatable in vitro and infectious to protoplasts and plants.
3. To study subdomain organization of the movement protein (MP) encoded by the first gene in the triple gene block (TGBp1), we mutated the 25-kDa TGBp1 of the potato virus X (PVX). 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 green fluorescent protein with 25-kDa MP and its derivative lacking motifs V - VI exhibited similar fluorescence pattern 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 (PlAMV), but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV 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.
4. We have shown that the 75-nt untranslated region (5’-UTR) upstream of the MP-gene of crTMV RNA (IRESCR MP75) was able to promote internal initiation of translation of the 3’-proximal reporter genes from the synthetic dicistronic RNA transcripts. Moreover, the equivalent sequence from TMV U1 RNA used as the intercistronic spacer (IRESU1 MP75) was able of mediating internal translation of the second gene in dicistronic transcripts. Both of these sequences were nonfunctional as IRESs within full-length RNAs: not unexpectedly the MP could not be translated in vitro from genomic RNAs. Furthermore, the dicistronic RNA transcripts structurally equivalent to I2 sgRNA's of crTMV and TMV U1 but containing a stable 5’-terminal hairpin immediately upstream of the untranslated IRESMP75 sequence were able to express the MP gene. These results indicate that the 75-nt 5’-UTRS of crTMV and TMV UI positioned between a RNA hairpin and MP gene directed internal initiation of sgRNA I2 translation.
5. The beet yellows closterovirus (BYV)-encoded p65 protein, a homologue of HSP70 cell chaperones, was suggested to play a role of the virus movement protein (MP). To test this hypothesis, two types of complementation experiments were used, with the plant viruses containing the Triple Gene Block of MP genes. In one, the BYV p65 gene was cloned into a 35S promoter plasmid and introduced into Nicotiana benthamiana plants by microprojectile bombardment along with the 35S promoter-driven GUS-gene-tagged cDNA of a transport-deficient potexvirus mutant. Transient expression of the p65 complemented the mutant as visualised by the significant increase in the number of cells expressing the GUS reporter gene in the infection foci. In the other test, the p65 gene was inserted into the infectious cDNA of the hordeivirus RNAb component to replace either the 58 kDa MP gene or the whole TGB. Inoculation of Chenopodium quinoa and Ch. amaranticolor plants with the T7 transcripts of the chimeric RNA b , together with the hordeivirus RNAs a and g , caused symptomless infection in inoculated leaves detected by hybridization of the total leaf RNA with a specific cDNA probe. The ability of the BYV p65 to substitute for the potexvirus or hordeivirus MPs provides direct evidence for its involvement in the cell-to-cell movement of closterovirus infection. The experiments on mapping the minor capsid protein (p24) in the BYV particles showed that the p24 encapsidates the 5’-terminal region of the virus genome.
The experiments of mapping the minor capsid protein in BYV particles
(p24, a putative vector transmission determinant of closteroviruses) were
conducted. It was found that the p24 encapsidates the 5’-terminal region
of the viral genome.
Picornaviruses, small RNA-containing animal viruses, include causative agents of poliomyelitis, hepatitis A, foot and mouth disease etc. Some key problems of their molecular biology remain unsolved. Important information related to 3 aspects has been accumulated upon implementation of the project. (a) New relationships between control elements of the 5'-noncoding region of the viral RNA and biological properties (e. g., neurovirulence) of picornaviruses were elucidated. In particular, it was shown that point mutations in the translational starting window of encephalomyelitis virus RNA may not only lower neurovirulence but - at an equal level of attenuation- also critically affect the clinical pattern of the disease. The growth of the mutant viruses in the standard cell cultures may remain unchanged. These and other observations made suggest the existence of tissue-specific peculiarities of the translation machinery and also point out to the necessity of new approaches to the virus identification in molecular epidemiology and clinical practice. (b) The cis-element, named oriR, involved in the initiation of the (-) strand of enterovirus (poliovirus and coxsackievirus) RNA was characterized. OriR was demonstrated to possess a multidomain quasi-globular structure maintained by tertiary ("kissing") interactions between the loops of RNA hairpins. Destabilization of the kissing interaction resulted in the impairment of the oriR function. (c) Nonreplicative recombination between viral RNA genome fragments was demonstrated for the first time. Nonviable (nontranslatable and nonreplicable) fragments of poliovirus RNA can join each other, generating a bona fide viral genome. Cryptic (possessing a negligible activity) ribozymes present in any natural high molecular mass RNA species may perhaps be involved in the nonreplicative RNA recombination. It is proposed that rare nonreplicative recombination events may be of significant evolutionary importance, resulting in the creation of novel viral genomes and possibly also cellular genes. Using the approach worked out, it becomes possible to study very inefficient RNA reactions yielding so little products that they cannot be detected by conventional techniques. However, these products, being represented by viral genomes, are able to self-multiplication and therefore can readily be recognized and accumulated in quantity.
The mutations at silent (synonymous, not changing protein sequence)
sites in codons disclose much about the features of molecular evolution.
An analysis of silent mutation fixation rates in nucleotide sequences encoding
VP1, 2A, 2B and 3D proteins of poliovirus type 1has been carried out. The
data set included sequences of wild strains representing an evolutionary
lineage and vaccine-derived isolates from a poliomyelitis patient with
common variable immunodeficiency syndrome. The results indicated that evolution
of VP1 and 3D sequences in the both lineages occurred at significantly
lower rates in comparison with that of 2A and 2B sequences. The rate of
synonymous substitution fixation in the 2A/2B sequences belonging to the
immunodeficient patient lineage appeared to be among the highest ever reported
for any of the RNA virus genes. The least favored or "rare" synonymous
codons seemed to be unexpectedly often conserved in polioviruses both related
and unrelated to each other. There are three times as many conserved rare
codons in the VP1 region than in the 2A/2B. The local synonymous codon
usage bias towards rare codons may be a result of selective constraints
imposed by important RNA secondary structure elements or by cotranslational
protein folding when the introduction of a "slow" codon alters the kinetics
of translation and ensures the proper folding of a nascent polypeptide.
In TP experiments with the intact viruses it was found that ts21-66 CP (158 aminoacid residues) is more accessible for hot tritium atoms than U1 CP. Total incorporation of stable tritium label into mutant CP was twice as high as that in U1 CP. This increase in labeling was largely determined by higher labeling of the most externally located C-terminal region of CP subunit. C-terminal segment (residues 149-158) incorporates largest amount of the label even in the case of U1 CP, but for ts21-66 CP it was almost three times higher. At the same time, the label incorporation into 62-68 loop (also located close to the virion surface) for ts21-66 was two times lower than for U1. From this data it may be concluded that the aminoacid substitutions I21® T and D66® G induce significant changes in tertiary structure of surface-located regions of TMV CP molecule. This transition results in a further increase in accessibility of the C-terminal segment of TMV CP molecule and in partial masking of the 62-68 loop (probably by movement of C-terminal segment).
Also in 1998, in collaboration with V.A.Drachev laboratory we have undertaken a comparative study of thermostability of U1 and ts21-66 CPs by differential scanning calorimetry. It was observed that free TMV CP molecules display surprisingly low thermal stability: denaturation temperatures of wild type and mutant CP subunits equaled 41 and 38 deg. C respectively. Incorporation of CP subunits into TMV virions resulted in large increase in thermal stability. In this case denaturation temperatures turned out to be the same (72 deg.) for both U1 and ts21-66.This means that high thermal stability of TMV virions is determined largely by INTERsubunit protein-protein interactions and by RNA-protein interactions.
Our main goal is the isolation, structural analysis and functional study of the virus and cell covalent complexes of nucleic acids and proteins. Mechanisms of «Formation-Disruption» of the complexes are also under consideration.
In this connection, a study of the unlinking enzyme which splits off VPg from picornaviral RNAs, is in a progress. In order to study, in part, a role of the unlinking enzyme in picornavirus outgrowth, immunochemical analysis and phase-contrast, immunofluorescent and atomic force microscopies are used. To this purpose antisera against EMC virus VPg-RNA were raised and then IgGs specific to the covalent linkage unit (CLU) were identified.
In the course of this research, we discovered that antibodies raised
against virus RNAs reveal specificity to the selective antigenic structures
(ribotopes) and recognize the RNA antigens variously. Nowadays, to prove
a specificity of the antibodies above, a new method was developed, that
is an atomic force microscopy of the virus RNA and ribonucleoproteins.
We are also engaged in a non-radioactive detection of the virus nucleic
acids and viroids for diagnostic purposes.
Motogens are the special type of agents, which can influence cell shape and motility. Such action is essential for embryonic development, play important role in different disease - tumour invasion, wound healing. We wished to find out what is the effect of the motogens, scatter factor (HGF/SF) and tumour promoter, phorbol ester (PMA), on the multinuclear cells. The multinuclear cells are the super-coherent structure, where the territories of individual cells are not only linked by cell-cell contacts but also united in the same cytoplasm. Using the videoenchanced microscopy we have shown that after HGF\SF or PMA treatment the cytoplasm of multinuclear cells segregated into several lamellar domains - lamelloplasts connected with each other by cylindrical cables. Such segregation was accompanied by significant reorganisation of the whole system of cytoskeleton, redistribution of nuclei and centrosomes. Lamelloplasts, especially small ones, were highly motile, contained from 0 to several nuclei and at least one centrosome. In contrast cables had no nuclei and centrosomes and had no motility of their own. It was shown that normal organisation of microtubular system is essential for segregation. Thus in these experiments we have discovered new phenomenon: segregation of cell cytoplasm into two types of domains. Such ability for segregation may be a fundamental property of mononuclear and multinuclear cells used in various situations for many physiological purposes.
The intercellular interactions are very important for the tissue organisation. We used the models with multinuclear cells to investigate the role of focal contacts during cell-cell interaction. As a result of formation of multinuclear cells the size and amount of focal contacts were increased. So we have developed good model system, where cells of the same origin had different amount of focal contacts.
Our next task was to investigate the role of molecular composition of actin cytoskeleton on cell behaviour. It was recently shown that amount of cells, positive with alpha-smooth muscle actin, the special cytoskeletal protein, are increased during wound reparation, fibrocontractive diseases, or stroma reaction to epithelial tumours. ASM-positive and ASM-negative fibroblasts were significantly different, independently of their origin. We had shown that ASM-positive cells had larger average areas, higher numbers of narrow extensions at the edges, larger focal adhesions with the substratum and a more important network of cellular fibronectin than ASM-negative cells. So it was shown that presence of ASM-actin in microfilaments leads to appearance of strong differences in cell morphology and cell contact structures and, probably have the special functional importance and associated with distinct functional activities.
The Project aimed at two subjects of investigation:
I. The study of gap junction intercellular communication and adhesive contacts between normal and tumor cells as a two-factor complex in the cell growth control.
A new experimental model for cultivation of the tumor and normal tissues extirpated from adenocarcinoma of the human large intestine have been developed. It allows to study the permeability of the intercellular gap junctions (GJ) in cell growth zones developing from the explants of these tissues on the transparent collagen base layer. Dynamics of growth zone formation around the normal and tumor explants (NE and TE) have been studied. Morphological analysis of the main types of the cells forming growth zone of NE and TE has been carried out accordingly. Computer registration system for videoimages of the cell culture has been used for study of the permeability of the GJs by fluorescent dye transfer technique.
II. The mathematical models of cell growth.
An approach for description of interaction between cancer cells, normal cells and those of the immune system is suggested. The approach helps to describe spacial effects of the interaction. For this purpose it is suggested to use an indicator of cell fractal dimension. A model of cancer development (a system of differential equation) has been made. There are several types of model functioning. It was demonstrated that an equation type for cancer fractal dimension change determines the following types of the model: the existence of a small number of cancer cells, unlimited proliferation development and fluctuation of cancer cells number. The analysis of cancer spreading has been conducted. Determinations of cancer spreading by age and sex have been found. A model of free competition describing cancer spreading is suggested.
Experimental systems have been developed to study functional properties and structure of tight junctions (TJ). Three sub-lines (MDCK-1, MDCK-2, MDCK-N) of the MDCK cell line were tested under various cell culture conditions. Cells MDCK-N grown on collagen substrate were chosen for the further experiments, as these cells formed tight continuos monolayer without cell-free areas. Intercellular transfer of membrane-soluble molecules was studied on this cell system using fluorescent lipid probes C6-NBD, R18, PKH-26 and perylene-labeled PC (PPC). The following loading protocols were used: (1) plating of the cell suspension containing a known fraction (1-10%) of the pre-labeled cells; (2) plating of the suspended pre-labeled cells on the monolayer of the non-labeled cells; (3) local labeling of a cell in a monolayer using patch-pipette in a cell-attached configuration; (4) labeling of a cell monolayer and subsequent photobleaching of a small area (FRAP). Experiments with local labeling with PPC revealed intercellular transfer of the lipid probe in a monolayer of MDCK-N cells. This result favors the lipid model of TJ suggesting a lipidic continuity in TJ. For the measurements of transepithelial resistance (TER) the cells were grown on collagen membranes (Cellagen). TER was measured in MDCK-N cells under control conditions and effects of some known modulators of TJ (low-Ca medium, temperature) were studied.
We have applied a methodology, called SEREX (serological identification of antigens by recombinant expression cloning) to search for tumor antigens in two types of cancer. This approach is based on the known property of many tumors to elicit a humoral immune response. Renal cell carcinoma and breast carcinoma were used to prepare a cDNA expression libraries, which were then screened with autologous sera. In case of renal cancer 51 positive clones, and in case of breast cancer 49 positive clones were isolated. By partial sequencing of their cDNA inserts, we have identified 54 clones as corresponding to 36 known genes. Other sequences are absent from public databases. The positive clones were then screened with a panel of allogeneic patients' and normal sera. In case of renal cancer two clones have been selected, which show reactivity with sera of cancer patients and do not react with about 20 normal sera. Both of them, REN-46 and REN-54, are novel genes. Similarly, in the case of breast cancer two genes have been identified, BC-416 and BC-203. BC-416 is a recently discovered cxorf5 gene of unknown function; BC-203 is novel gene. At the moment, these four genes are being considered as candidate tumor antigens. Additionally, SEREX analysis has identified a large number of known and unknown autoantigens, some of them apoptosis-related proteins.
Cytokines of Tumor Necrosis Factor (TNF) family can signal activation, proliferation and apoptosis of cells of various lineages. In vitro TNF and lymphotoxin (LTa), a close relative of TNF, can bind and signal as homotrimers via TNF receptors. Additionally, LT (in the form of LTa/LTb heterotrimer), but not TNF, can signal through separate receptor, called LTbR. Knock-out models of the first generation with single targeted TNF, LTa, LTb, TNFRp55 and LTbR genes revealed the major role of these two signaling pathways in the development and maintenance of functional microarchitecture of the peripheral lymphoid tissues. We now applied the Cre-loxP technology to generate mice with tissue-specific and/or inducible inactivation of TNF or LT genes. To this end, three genetic constructs were prepared and used to target each of the three genes separately, and the neo markers were eliminated from the ES cells by additional transfections with Cre-expressing vector. As a result, mice have been generated which contain single TNF or LT genes flanked by loxP sites and thus targeted for subsequent Cre-mediated deletion in vivo. These mice are being crossed to several tissue-specific Cre transgenic mice to address the role of TNF and LT expressed by distinct cell types in vivo.
Transmitters and mechanisms involved in motor control in molluscs are investigated. Comparative studies of the pond snail Lymnaea stagnalis which crawls due to the ciliated epithelium covering the sole of the foot and the land snail Helix lucorum using pedal muscular waves for locomotion have revealed similar fundamental mechanisms controlling locomotory speed. 1) Both snails varied arbitrarily and significantly the length of foot sole during crawling and mollusc speed correlated directly with sole length (Pavlova, 1990, 1994). 2) Serotonin (5-HT) switched on locomotion, increased considerably locomotory speed, but did not change values of sole length (Pavlova, 1996, 1997). Thus the linear dependence between sole length and locomotory speed was shifted up along the axis of locomotory speed. In Helix, that shift was provided by an increase in the number of muscle cells involved in muscular waves. 3) Ergometrine, the blocker of inhibitory dopamine receptors, switched on locomotion, elongated foot sole and increased proportionally locomotory speed (Pavlova, submitted). In crawling Helix, during sole contraction and elongation, the number of muscle cells in the waves kept constant and locomotory speed variability (increase/decrease) was provided by proportional change in the contraction amplitude of muscle cells involved in muscular waves. We found that in Lymnaea, smooth muscle cells situated just under the ciliated epithelium of the sole contained giant mitochondria (Bakeeva, et al., 1992). Smooth muscle cells located under sole ciliated epithelium in Helix had the same structure (Rogers, 1969).
All data mentioned above allow to suggest fundamental mechanisms controlling locomotory speed in wide range in molluscs varying arbitrarily and considerably the length of foot during locomotion (Pavlova, submitted). Serotonin switches on rhythmic locomotor generator (muscular wave generation with constant frequency). The concentration of 5-HT in the CNS and target sensitivity to 5-HT depend on season and physiological state of animal (Dyakonova, 1988; Lent et al., 1991). We consider that according to the season and the state of animal, 5-HT determines the number of muscle cells involved in muscular waves for each locomotor episode and shifts the linear dependence between sole length and locomotory speed along the axis of locomotory speed. During locomotor episode dopamine and some unknown transmitter control the locomotory speed in wide range via sole length change and the modulation of contraction amplitude of muscle cells involved in muscular waves. Dopamine contracts the sole and decreases the contraction amplitude respectively, the unknown transmitter elongates the sole and increases the contraction amplitude proportionally. The balance of the two transmitters shifts the corresponding point along the plot of linear dependence between sole length and locomotory speed. Thus mollusc can efficiently control its speed changing the only one parameter - the foot length.
We expect the unknown transmitter belong to peptide family discovered recently in the CNS of Aplysia (Lloyd & Connolly, 1989) and Tritonia (Lloyd et al., 1996). We showed that Tritonia peptide increased locomotor activity in the mollusc (Willows et al., 1997, 1998). Using antisera directed towards Tritonia peptide in Lymnaea, we found immunoreactive neurons in the pedal ganglia and immunolabelled nervous fibers in the foot.
In contrast to molluscan locomotor system where both transmitter 5-HT and the peptide increased motor activity, in other systems the transmitters caused opposite effects. We found that in Tritonia the peptide increased and 5-HT unexpectedly inhibited esophageal ciliar activity (Pavlova et al., in press).
To describe electrical phenomena observed in growth of Neurospora crassa hyphae, a theoretical model was developed considering the hyphae as a one-dimensional electric cable with non-uniform longitudinal distribution of current sources. Theoretical analysis of the experimental data in the framework of this model allows to take quantitative estimations of the energy cooperation between apical and distal parts of the growing hyphae. In this case the appropriated membrane potential level of apical cells is supported through the intercellular electrical currents propagation generated by the proton pumps consuming the ATP resources at the distances of 300-1000 mkm from the hyphal tip.
The method of semi-automatic computer analysis has been elaborated for the growth hyphal tip images. Combining this method with the novel experimental device allowed to isolate apical hyphal fragment with the minimal traumatic stress, we have distinguished three principal types of mycelial tree self-organization based on: (i) individual module parameters (300-400 mkm); (ii) energy cooperation at the distances of spatial constant of equivalent electric cable (700-800 mkm); (iii) axial metabolite transport (upto 6000 mkm from the tip).
Comparative analysis of the mutant N.crassa strain growth puts forward the problem to distinguish between genetic and epigenetic modes to operate by the self-organization of fungal mycelium as a multimodule system.
The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors Suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolysed added ATP and this reaction was inhibited by Suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM.
We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP-receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.
Suramin is a well known anticancer agent, and the clinical trial of suramin revealed some side effects, including neutropenia with a drop of up to 90% of the blood neutrophil count in a majority of patients. The mechanism of suramin-induced neutropenia is unknown. Our results on adhesion may explain neutropenia observed under suramin treatment and may be connected with inhibition of ecto-ATPase by suramin.
Human polymorphonuclear leukocytes (PMN) were found to tightly adhere on endothelial (lines EAhy926 and ECV304) and collagen surfaces under the influence of suramin. This was observed by scanning electron microscopy. Parallel to adhesion PMN 5-lipoxygenase (5-LO) was inhibited after stimulation with the Ca-ionophore A23187. Suramin decreased the release of radiolabeled arachidonic acid (AA) and 5-LO metabolites by prelabeled PMNs stimulated with A23187. Using agents, releasing the suramin-stimulated adhesion, jasplakinolide and dextran sulfate, we observed reversal of suramin effect on leukotriene synthesis. Jasplakinolide released the adhesion of PMN on endothelial and collagen-coated surfaces and restored 5-LO activity. Dextran-sulfate released adhesion on collagen-coated surface and abolished suramin inhibition. Arachidonate could also overcome adhesion and inhibition of 5-LO. We conclude that suramin-induced tight attachment of PMN on to solid surface lead to decreased leukotriene synthesis during subsequent A23187 stimulation in the absence of exogenous substrate.
We elaborated a technique for isolation and purification of a highly enriched fraction of the centromeric heterochromatin (chromocentres) from mouse hepatocytes and invented methodological assays to control purity of the fraction. They were based on the electron microscopy and Western blot analysis of the nucleolar contaminations using antibodies to specific nucleolar proteins. By in situ immunolabeling of isolated chromocentres, we showed that they contained anti-kinetochore proteins CENP-A, B and C. By electrophoresis, fractions were demonstrated to be enriched in histones and two nonhistone proteins with Mr 50-70 kD. Western blots also confirmed that isolated chromocentres contained CENP-A protein. In order to describe a spatial distribution of chromocentres within nuclei, a computer image processing software was developed and applied for the 3D studies of chromocentres and kinetochores during the cell cycle in 3T3NIH mouse fibroblasts. For the first time we showed that chromocentres and kinetochores remained structurally connected throughout the whole interphase. We finished the electron microscopic analysis of the centromeric complex in human mitotic cells infected with cytomegalovirus (CMV). The results obtained indicated that CMV blocked the assembly of characteristic (threelayers) kinetochores, that might be a reason of cell arrest at metaphase. Based on these results, we supposed that CMV induced premature condensation of chromosomes similar to that induced by okadaic acid, a potent phosphatase 1 and phosphatase 2A inhibitor.
Ultrastructure of intermitochondrial junctions was investigated in
rat cardiomyocytes at various stages of alcoholic cardiomyopathy. The progress
of gradually developing structural changes depends on the stage of the
disease. The first stage is characterized partial reorganization of intermitochondrial
junctions. In late stage intermitochondrial junctions disappear and destructive
irreversible changes develop in the ultrastructural organization of mitochondria.
These changes of intermitochondrial junction may be considered as a sign
of impairment of myocardial functioning. The structure of intermitochondrial
junctions was studied using fixation of cardiac tissue with addition of
colloidal lantanum. Analysis of ultrastructure intermitochondrial junctions
was based on computer-assisted analyses of serial electron micrographs.
We have shown, that intermitochondrial junction is not the result of tight
mitochondrial adjournment. They are special membrane structures with special
organization, like intercellular junctions.
The method of tritium planigraphy has been used to resolve the three-dimensional structure of a protein by identifying residues accessible for labeling. The semiempirical algorithm proposed for globular proteins involves (i) theoretical prediction of the secondary structure elements (SSEs), (ii) experimental determination of the residue-accessibility profile by bombarding the whole protein with a beam of hot tritium atoms, (iii) estimation of the residue-accessibility profiles for isolated SSEs by computer simulation, (iv) location of contacts between SSEs by collating the experimental and simulated accessibility profiles, and (v) assembling of SSEs into a compact model via these contact regions in accordance with certain rules. For sperm whale myoglobin, carp and pike parvalbumins, the l cro repressor, and hen egg lysozyme, this algorithm yields the most realistic models when SSEs are assembled sequentially from the amino to the carboxyl end of the protein chain.
Intact influenza A virions were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the matrix protein M1 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 M1 fragment 2-158 [Sha, B. And Luo, M. (1997) Nature Struct. Biol. 4, 239-244].
GeneBee is the national bioinformatic facility for Russian Scientists, organized by Belozersky Institute. As EMBnet node it became operational since 1996. The functions of GeneBee are the following:
GeneBee provides molecular biology databases and software facilities for over 90 departments and laboratories within Russian Federation.
GeneBee participates in the project "Means of maintenance of researches on physics-chemical biology and biotechnology in 1992-2005 years" (chairman of Advice Prof. D.G.Knorre).
Within the framework of the "Human Genome" program GeneBee group
carries out a support in biocomputing to the following organizations: