M.V.
Lomonosov Moscow State University
ANNUAL REPORT
2002
CONTENT
Bioenergetics
and photosynthesis
Mathematical models in biology
Structure, expression and evolution of genom
I. BIOLOGY OF CELL AND CELL
ORGANELLES
Deliagina T.G., Pavlova E.L.
Journal of Neurophysiology 87 (2002) 1-14
A postural
control system in the lamprey is driven by vestibular input and maintains the
dorsal-side-up orientation of the animal during swimming. After a unilateral
labyrinthectomy (UL), the lamprey continuously rolls toward the damaged side.
Normally, a recovery of postural equilibrium ("vestibular compensation")
takes about 1 mo. However, illumination of the eye contralateral to UL results
in an immediate and reversible restoration of equilibrium. Here we used eye
illumination as a tool to examine a functional recovery of the postural
network. Important elements of this network are the reticulospinal (RS)
neurons, which are driven by vestibular input and transmit commands for
postural corrections to the spinal cord. In this study, we characterized
modifications of the vestibular responses in individual RS neurons caused by UL
and the effect exerted on these responses by eye illumination. The activity of
RS neurons was recorded from their axons in the spinal cord by chronically
implanted electrodes, and spikes in individual axons were extracted from the
population activity signals. The same neurons were recorded both before and
after UL. Vestibular stimulation (rotation in the roll plane through 360
degrees ) and eye illumination were performed in quiescent animals. It was
found that the vestibular responses on the UL-side changed only slightly,
whereas the responses on the opposite side disappeared almost completely. This
asymmetry in the bilateral activity of RS neurons is the most likely cause for
the loss of equilibrium in UL animals. Illumination of the eye contralateral to
UL resulted, first, in a restoration of vestibular responses in the neurons
inactivated by UL and in an appearance of vestibular responses in some other
neurons that did not respond to vestibular input before UL. These responses had
directional sensitivity and zones of spatial sensitivity similar to those
observed before UL. However, their magnitude was smaller than before UL.
Second, the eye illumination caused a reduction of the magnitude of vestibular
responses on the UL side. These two factors tend to restore symmetry in
bilateral activity of RS neurons, which is the most likely cause for the
recovery of equilibrium in the swimming UL lamprey. Results of this study are
discussed in relation to the model of the roll control system proposed in our
previous studies as well as in relation to the vestibular compensation.
Dergunova N.N., Bulycheva T.I., Artemenko E.G., Shpakova A.P., Pegova
A.N., Gemjian E.G., Dudnik O.A., Zatsepina O.V., Malashenko O.S.
Immunology Letters 83 (2002) 67-72
A novel
monoclonal antibody (Mab) (called 3C9) against a major nucleolar phosphoprotein
B23 was used to study 1323 qualitative and quantitative alterations in phytohemagglutinin
(PHA) - stimulated human peripheral blood lymphocytes in indirect
immunofluorescence and Western blots. It was shown that lymphocyte
proliferation was accompanied by gradual augmentation of nucleoli and their
accumulation of the protein B23 tip to 2-fold by 16 h and 40-50 fold by 72 h.
as compared with the non-stimulated cells. By parallel immunolabeling with the
anti-Ki-67 antibody. it was shown that the early changes of 1323 amount and
localization occurred before an appearance of Ki-67 protein, a ell-known marker
of proliferating cells. Our results evidence that antibodies against B23 might
be applied for recognition of human peripheral lymphocytes at early stages of
their activation for proliferation, preceding the S-phase.
Dunina-Barkovskaya A.Y., Bujurina I.M., Pivovarov V.S., Frolov V.A.
Membrane and Cell Biology 14 (2001) 791-811
Weak acids are
efficient blockers of gap-junctional conductance. It is generally accepted that
intracellular acidification produced by weak acids fully accounts for the
gap-junctional uncoupling. Protonation of the cytoplasmic portions of the
channel-forming protein connexin is thought to lead to the conformational
changes switching the channel from the open into the closed state. If this is
the only mechanism of the weak-acid induced uncoupling, then the correlation
between junctional conductance (Gj) and intracellular pH (pHin)
should not depend on the means of intracellular acidification. We compared the
responses of junctional conductance in BHK cells measured in double whole-cell
experiments to the applied transmembrane concentration gradients of bicarbonate
or ammonium. These treatments were to lower pHin in a predictable
way according to the equations: pHin = pHout -lg[[HCO3]out/HCO3-]in)
or pHin = PHout - lg[[NH4+]in[NH4+]out),
respectively. We found that the behavior of Gj depended on the substance used.
At a 500-fold bicarbonate gradient (calculated pHin approximately
4.8) the cells remained coupled, while a 100- or 10-fold gradient of ammonium
imposing pHin approximately 6.1 produced fast uncoupling. The
responses of junctional conductance were often accompanied or preceded by
changes of non-junctional membrane conductance. We suggest that the mechanisms
of the weak acid/base-induced channel gating may contain an additional
"lipophilic" component due to the presence of the non-dissociated
form of the acid/base in cell membrane.
Fetisova E.K., Ivanova O.Yu., Omelchenko T., Vasiliev Ju.M.
Russian Journal of Developmental Biology 33 (2002) 306-310
We studied the
interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed
culture. Heterotypic cells had a different cytoskeleton organization and
expressed different cell adhesion molecules, cadherins. In spite of this, when
the cells contacted in the mixed cultures, a heterophilic contact was formed
and the actin cytoskeleton of an epitheliocyte at the site of contact was
reorganized: the marginal actin bundle was decomposed and actin structures were
formed in its place, that were typical for the fibroblast lamella. No changes
were observed in the actin organization of the fibroblast. In architecture, the
heterophilic adhesion contacts resembled the contacts between fibroblasts. Both
heterophilic and homophilic contacts were transient, rather than constant
structures. The formation of heterophilic contacts in mixed cultures can serve
as a model of formation of a tissue system consisting of epithelium and
mesenchyme.
Mode of epithelium-fibroblast
interaction in mixed heterotypic cell cultures
Fetisova E.K., Ivanova O.Iu., Vasil'ev Iu.M.
Tsitologiia 44 (2002) 235-241
The interaction
between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts
(diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic
cell cultures. The mode of cell interaction depends on the manner of their
collision. At collision of the epithelium lamella and the lateral side of
fibroblast, the lamella was seen to creep under the lateral side to force back
the fibroblast. At the frontal collision of epithelium and fibroblast lamellae,
the mode of interaction depends on the local situation. With the presence of a
free substratum around, the fibroblast formed a new lamella and moved aside
from the place of collision. In the case, when the neighboring cells prevented
fibroblast from moving, it migrated under the epithelium. In this work, we have
first demonstrated the formation of specialized intercellular adhesions between
epithelium and fibroblasts. The cultures were studied by phase contrast,
interference reflection or video tape recording, using an image processing
system (Hamamatsu). For studying adhesion, immuno-fluorescent methods were
performed.
Kapinya K.J., Lowl D., Futterer C., Maurer M., Waschke K.F., Isaev N.K.,
Dirnagl U.
Stroke 33 (2002) 1889-1898
Background and
Purpose-We tested whether volatile anesthetics induce neuroprotection that is
maintained for a prolonged time. Methods-Rats were pretreated for 3 hours with
1 minimal anesthetic concentration of isoflurane or halothane in normal air
(anesthetic preconditioning [AP]). The animals were subjected to permanent
middle cerebral artery occlusion (MCAO) at 0, 12, 24, or 48 hours after AP.
Halothane-pretreated animals were subjected to MCAO 24 hours after AP.
Histological evaluation of infarct volumes was performed 4 days after MCAO.
Cerebral glucose utilization was measured 24 hours after AP with isoflurane.
Primary cortical neuronal cultures were exposed to 1.4% isoflurane for 3 hours.
Oxygen-glucose deprivation (OGD) was performed 24 hours after AP. Injury was
assessed 24 hours later by measuring the release of lactate dehydrogenase into
the medium 24 hours after OGD. Results- Isoflurane anesthesia at 0, 12, and 24
hours before MCAO or halothane anesthesia 24 hours before MCAO significantly
reduced infarct volumes (125 ± 42 mm3, P=0.024; 118 ± 51 mm3, P=0.008;
120 ± 49 mm3,
P=0.009; and 121 ± 48 mm3, P=0.018, respectively) compared with control volumes
(180 ± 51 mm3).
Three hours of isoflurane anesthesia in rats did not have any effect on local
or mean cerebral glucose utilization measured 24 hours later. Western blot
analysis from cortical extracts of AP-treated animals revealed an increase of
the inducible NO synthase (iNOS) protein beginning 6 hours after AP. The iNOS
inhibitor aminoguanidine (200 mg/kg IP) eliminated the infarct-sparing effect
of AP. In cultured cortical neurons, isoflurane exposure 24 hours before OGD
decreased the OGD- induced release of lactate dehydrogenase by 49% (P=0.002).
Conclusions-Pretreatment with volatile anesthetics induces prolonged
neuroprotection in vitroand in vivo, a process in which iNOS
seems to be critically involved.
Komarova Y.A., Vorobjev I.A., Borisy G.G.
Journal of Cell Science 115 (2002) 3527-3539
Microtubule dynamics
were investigated in CHO and NRK cells by novel experimental approaches
designed to evaluate the microtubule behavior in the cell interior. These,
approaches were: (1) laser photobleaching of a path through the centrosome; (2)
direct observation of microtubules in centrosome-containing cytoplasts; (3)
GFP-CLIP-170 expression as a marker for microtubule plus end growth; and (iv)
sequential subtraction analysis. The combination of these approaches allowed us
to obtain data where the density of microtubules had previously prevented
conventional methods to be applicable. In the steady state, nascent
microtubules grew persistently from the centrosome towards the cell margin.
Frequently, they arrived at the cell margin without undergoing any transition to
the shortening phase. In contrast to the growth of microtubules, shortening of
the plus ends from the periphery was non-persistent; that is, rescue was
frequent and the extent of shortening showed a distribution of lengths
reflecting a stochastic process. The combination of persistent growth and a
cell boundary led to a difference in apparent microtubule behavior in the cell
interior compared with that near the cell margin. Whereas microtubules in the
cell interior showed asymmetric transition frequencies, their behavior near the
cell margin showed frequent fluctuations between phases of shortening and
growth. Complete microtubule turnover was accomplished by the relatively rare
episodes of shortening back to the centrosome. Release from the centrosome with
subsequent minus end shortening also occurred but was a minor mechanism for
microtubule turnover compared with the plus end pathway. We propose a life
cycle for a microtubule which consists of rapid growth from the centrosome to
the cell margin followed by an indefinite period of fluctuations of phases of
shortening and growth. We suggest that persistent growth and asymmetric
transition frequencies serve the biological function of providing a mechanism
by which microtubules may rapidly accommodate to the changing shape and
advancing edge of motile cells.
Levina N.N., Dunina-Barkovskaya A.Y., Shabala S., Lew R.R.
Journal of Membrane Biology 188 (2002) 213-222
Blue light is the
primary entrainment signal for a number of developmental and morphological
processes in the lower eucaryote Neurospora crassa. Blue light regulates
photoactivation of carotenoid synthesis, conidiation, phototropism of
perithecia and circadian rhythms. Changes in the electrical properties of the
plasma membrane are one of the fastest responses to blue light irradiation. To
enable patch- clamp studies on light-induced ion channel activity, the wall-
less slime mutant was used. Patch-clamp experiments were complemented by
non-invasive ion-selective measurements of light-induced ion fluexes of slime
cells using the vibrating probe technique. Blue light usually caused a decrease
in conductance within 2-5 minutes at both negative and positive voltages, and a
negative shift in the reversal potential in whole-cell patch-clamp
measurements. Both K+ and Cl- channels contribute to the
inward and outward currents, based on the effects of TEA (10 mm) and DIDS
(500 ?m). However, the negative shift in
the reversal potential indicates that under blue light the Cl-
conductance becomes dominant in the electrical properties of the slime cells
due to a decrease of K+ conductance. The ion-selective probe
revealed that blue light induced the following changes in the net ion fluxes
within 5 minutes: 1) decrease in H+ influx; 2) increase in K+
efflux; and 3) increase in Cl- influx. Ca2+ flux was
unchanged. Therefore, blue light regulates an ensemble of transport processes:
H+, Cl-, and K+ transport.
Nikolaeva O.P., Orlov V.N., Bobkov A.A., Levitsky D.I.
European Journal of Biochemistry 269 (2002) 5678-5688
The thermal
unfolding of myosin subfragment 1 (S1) cleaved by trypsin was studied by
differential scanning calorimetry. In the absence of nucleotides, trypsin
splits the S1 heavy chain into three fragments (25, 50, and 20 kDa). This
cleavage has no appreciable influence on the thermal unfolding of S1 examined
in the presence of ADP, in the ternary complexes of S1 with ADP and phosphate
analogs, such as orthovanadate (Vi) or beryllium fluoride (BeFx), and in the
presence of F-actin. In the presence of ATP and in the complexes S1.ADP.Vi or
S1.ADP.BeFx, trypsin produces two additional cleavages in the S1 heavy chain: a
faster cleavage in the N-terminal region between Arg23 and Ile24, and a slower
cleavage at the 50 kDa fragment. It has been shown that the N-terminal cleavage
strongly decreases the thermal stability of S1 by shifting the maximum of its
thermal transition by about 70C to a lower temperature, from 500C
to 42.40C, whereas the cleavage at both these sites causes dramatic
destabilization of the S1 molecule leading to total loss of its thermal transition.
Our results show that S1 with ATP-induced N-terminal cleavage is able, like
uncleaved S1, to undergo global structural changes in forming the stable
ternary complexes with ADP and Pi analogs (Vi, BeFx). These changes are
reflected in a pronounced increase of S1 thermal stability. However, S1 cleaved
by trypsin in the N-terminal region is unable, unlike S1, to undergo structural
changes induced by interaction with F-actin that are expressed in a 4-50C
shift of the S1 thermal transition to higher temperature. Thus, the cleavage
between Arg23 and Ile24 does not significantly affect nucleotide-induced
structural changes in the S1, but it prevents structural changes that occur
when S1 is bound to F-actin. The results suggest that the N-terminal region of
the S1 heavy chain plays an important role in structural stabilization of the
entire motor domain of the myosin head, and a long-distance communication
pathway may exist between this region and the actin-binding sites.
Omelchenko T., Vasiliev J.M., Gelfand I.M., Feder H.H., Bonder E.M.
Proc. Natl. Acad. Sci. USA 99 (2002) 10452-10457
Cultured
fibroblasts possess a characteristic polarized phenotype manifested by an
elongate cell body with an anterior lamella whose cell edge is divided into
protrusion-forming and inactive zones. Disruption of the fibroblast microtubule
cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile
activity and concomitant loss of structural polarity. The functional
relationship of myosin-driven contractile activity to loss of fibroblast
anterior-posterior polarity is unknown. To dissect the roles of microtubule
assembly and of Rho-dependent contractility on structural polarization of
cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with
the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase
inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree
of polarization by developing a highly elongate cell body with multiple narrow
processes extended from the edges of the cell. Treatment of fibroblasts with
nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid
or polygonal cells having broad, flattened lamellae that did not form long
lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not
display anterior-posterior polarization. When treated with Y-27632, the cells
acquired a polarized, elongate shape with narrow protrusions and wide lamellas.
Nocodazole alone or in combination with Y-27632 did not change the discoid
shape of epitheliocytes, however treatment with Y-27632 produced thinning of
the lamellar cytoplasm. We conclude that microtubules provide the necessary
framework for polarization of fibroblasts and epitheliocytes, whereas
Rho-regulated contractility modulates the degree of polarization of fibroblasts
and completely inhibits polarization in epitheliocytes.
Pavlova E.L., Deliagina T.G.
Journal of Neurophysiology 88 (2002) 1136-1146
In the swimming
lamprey, a postural control system maintains a definite orientation of the
animal's longitudinal axis in relation to the horizon (pitch angle). Operation
of this system is based on vestibular reflexes. Important elements of the
postural network are the reticulospinal (RS) neurons, which are driven by vestibular
input and transmit commands for postural corrections from the brain stem to the
spinal cord. Here we describe responses to vestibular stimulation (rotation of
the animal in the pitch plane) in RS neurons of intact lampreys. The activity
of neurons was recorded from their axons in the spinal cord by chronically
implanted arrays of macroelectrodes. From the multielectrode recordings of mass
activity, discharges in individual axons were extracted by means of a
spike-sorting program, and the axon position in the spinal cord and its
conduction velocity were determined. Vestibular stimulation was performed by
rotating the animal in steps of 45 degrees throughout 360 degrees or by
periodical "trapezoid" tilts between the nose-up and -down positions.
Typically, the RS neurons exhibited both dynamic responses (activity during
movement) and static responses (activity in a new sustained position). The
neurons were classified into two groups according to their pattern of response.
Group UP neurons responded preferentially to nose-up rotation with maximal
activity at 0-135 degrees up. Group DOWN neurons responded preferentially to
nose-down rotation with maximal activity at 0-135 degrees down. Neurons of the
two groups also differed in the position of their axons in the spinal cord and
axonal conduction velocity. An increase in water temperature, which presumably
causes a downward turn in swimming lampreys, affected the activity in the UP
and DOWN groups differently, so that the ratio UP responses to DOWN responses
increased. We suggest that the UP and DOWN groups mediate the opposing
vestibular reflexes and cause the downward and upward turns of the animal,
respectively. The lamprey will stabilize the orientation in the pitch plane at
which the effects of UP and DOWN groups are equal to each other. In addition to
the main test (rotation in the pitch plane), the animals were also tested by
rotation in the transverse (roll) plane. It was found that 22% of RS neurons
responding to pitch tilts also responded to roll tilts. The overlap between the
pitch and roll populations suggests that the RS pathways are partly shared by
the pitch and roll control systems.
Prass K., Ruscher K., Karsch M., Isaev N., Megow D., Priller J., Scharff
A., Dirnagl U., Meisel A.
Journal of Cerebral Blood Flow and Metabolism 22 (2002) 520-525
The widely
prescribed drug desferrioxamine is a known activator of the hypoxia-inducible
transcription factor I (HIF-1) and the subsequent transcription of
erythropoietin. In the brain. HIF-1 is a master switch of the transcriptional
response to hypoxia, whereas erythropoietin is a potent neuroprotectant. The
authors show that desferrioxamine dose-dependently and time-dependently induces
tolerance against focal cerebral ischemia in rats and mice, and against
oxygen-glucose deprivation in purified cortical neurons. Desferrioxamine
induced HIF-1 DNA binding and transcription of erythropoietin in vivo,
the temporal kinetics of which were congruent with tolerance induction.
Desferrioxamine is a promising drug for the induction of tolerance in humans
when ischemia can be anticipated.
Prusov A.N., Zatsepina O.V.
Biochemistry (Moscow) 67 (2002) 423-431
A new method
for isolation of the constitutive heterochromatin (chromocenters) from
interphase nuclei of mouse liver has been developed. This method allows
separation of chromocenters of different size. Chromocenter fractions are
essentially free of nucleoli and other contaminants. In contrast to nuclei and
nucleoli, the chromocenter fraction is characterized by simpler protein
composition, this fraction having a reduced number of proteins (especially high
molecular weight proteins). Chromocenters contain all histone fractions;
however, the relative proportion of histone H1 is lower and histone H3 is
higher than in the total nuclear chromatin. The amount of non- histone proteins
of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei
and nucleoli. The use of immunocytochemistry and immunoblotting methods
revealed the presence of the specific kinetochore component, CENP A protein.
This suggests tight association of some molecular kinetochore components with
chromocenters in the interphase.
Serganova I., Ksenzenko V., Serganov A., Meshcheryakova I., Pyatibratov
M., Vakhrusheva O., Metlina A., Fedorov O.
Journal of Bacteriology 184 (2002) 318-322
We have
determined the nucleotide sequence of a flagellin gene locus from the
haloalkaliphilic archaeon Natrialba magadii, identified the gene products among
proteins forming flagella, and demonstrated cotranscription of the genes. Based
on the sequence analysis we suggest that different regions of the genes might
have distinct evolutionary histories including possible genetic exchange with
bacterial flagellin genes.
Shchepina L.A., Popova E.N., Pletjushkina O.Y., Chernyak B.V.
Biochemistry (Moscow) 67 (2002) 222-226
The release of
cytochrome c from intermembrane space of mitochondria into cytosol is
one of the critical events in apoptotic cell death. The important
anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it
was shown that apoptosis and release of cytochrome c induced by
staurosporine or by tumor necrosis factor-a in He La cells were not affected by
inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers
(CCCP, DIN P) that decrease the membrane potential at the inner mitochondrial
membrane. The inhibitors of respiration and the uncouplers did not affect also
the anti-apoptotic activity of Bcl-2.
Sheval E.V., Polyakov V.Y.
Biologicheskie Membrany 19 (2002) 237-242
Interchromatin
compartment behaviour during cell permeabilization and extraction was analyzed
in human cultured cells (HeLa). In cells permeabilized with the buffer which
destabilized nuclear matrix the reduced staining (compare with nuclei with
stabilized nuclear matrix) of interchromatin space by toluidine blue was
observed. This suggested that a part of acidic proteins and/or RNA was
extracted from the interchromatin material. Using antibodies to a protein of
interchromatin material - PCNA, two population of this protein were observed.
The replication factory variant was present in the nuclear matrix fraction,
whereas the protein non- participated in replication and homogeneously
distributed in nucleoplasm was absent from the nuclear matrix. The
nucleoplasmic PCNA was extracted during permeabilization in the buffer, which
destabilized nuclear matrix. Thus, the phenomenon of reduced staining by
toluidine blue was, probably, due to extraction of non-matrix compounds of the
interchromatin material. The data of present work suggest that proteins
behaviour during nuclear matrix preparation do not depend exclusively on the
preparation procedure, but in part on the protein state.
Sheval E.V., Prusov A.N., Kireev I.I., Fais D., Polyakov V.Y.
Cell Biology International 26 (2002) 579-591
The method of
chromatin photo-stabilization by the action of visible light in the presence of
ethidium bromide was used for investigation of higher-level chromatin
structures in isolated nuclei. As a model we used rat hepatocyte nuclei
isolated in buffers which stabilized or destabilized nuclear matrix. Several
higher-level chromatin structures were visualized: 100 nm globules-chromomeres,
chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed
chromatin. All these structures were completely destroyed by 2 M NaCl
extraction independent of the matrix state, and DNA was extruded from the
residual nuclei (nuclear matrices) into a halo. These results show that nuclear
matrix proteins do not play the main role in the maintenance of higher-level
chromatin structures. Preliminary irradiation led to the reduction of the halo
width in the dose-dependent manner. In regions of condensed chromatin of
irradiated nucleoids there were discrete complexes consisting of DNA fibers
radiating from an electron- dense core and resembling the decondensed
chromomeres or the rosette-like structures. As shown by the analysis of
proteins bound to irradiated nuclei upon high-salt extraction, irradiation
presumably stabilized the non-histone proteins. These results suggest that in
interphase nuclei loop domains are folded into discrete higher-level chromatin
complexes (chromomeres). These complexes are possibly maintained by putative
non-histone proteins, which are extracted with high- salt buffers from
non-irradiated nuclei.
Smirnova O.Iu., Dudnik O.A., Zatsepina O.V.
Tsitologiia 44 (2002) 5-13
It is well
known that fibrillar centers (FC) constitute an essential structural component
of the active nucleolus in mammalian cells, yet their role in regulation of
ribosomal gene transcription still remains an open question. Here, we studied
the activity of endogenous RNA polymerase I upon partial and complete
unraveling of nucleoli and FCs. The pattern of BrUTP incorporation in nuclei of
hypotonically-treated cells was shown to be essentially the same as in the
control untreated cells. Moreover, the sites of BrUTP incorporation, which
revealed the active PNA polymerase I, were completely coincident with
UBF-binding sites. These observations allow to conclude that structural
integrity of FCs is not a prerequisite for maintenance of the active RNA
polymerase I transcriptional complex. When the action of hypotonic shock was
ceased and the cells were transferred to a complete cultural medium, the
swollen nucleoli recovered to the control state. Therefore it is possible to
conclude that none of the main morphological nucleolar counterparts, such as
FCs, dense fibrillar component or the pars granulosa, is responsible for the
maintenance of the nucleolar structural and functional integrity. A suggestion
is made that this role may be played by the nucleolar matrix associated with
the RNA polymerase I transcriptional complex.
Tsiavaliaris G., Fujita-Becker S., Batra R., Levitsky D.I., Kull F.J.,
Geeves M.A., Manstein D.J.
EMBO Reports 3 (2002) 1099-105
Dominant-negative
inhibition is a powerful genetic tool for the characterization of gene function
in vivo, based on the specific impairment of a gene product by the coexpression
of a mutant version of the same gene product. We describe the detailed
characterization of two myosin constructs containing either point mutations
F487A or F506G in the relay region. Dictyostelium cells transformed with F487A
or F506G myosin are unable to undergo processes that require myosin II
function, including fruiting-body formation, normal cytokinesis and growth in
suspension. Our results show that the dominant-negative inhibition of myosin
function is caused by disruption of the communication between active site and
lever arm, which blocks motor activity completely, and perturbation of the
communication between active site and actin-binding site, leading to an
approximately 100-fold increase in the mutants' affinity for actin in the
presence of ATP.
Vorob’eva O.V., Karyagina A.S., Volkov E.M., Viryasov M.B., Oretskaya
T.S., Kubareva E.A.
Russian Journal of Bioorganic Chemistry 28 (2002) 363-370
The functional
groups of the DNA methylation site that are involved in the DNA interaction
with methyltransferase SsoII at the recognition stage were identified. The
contacts in the enzyme–substrate complex were analyzed in the presence of
S-adenosyl-L-homocysteine using the interference footprinting assay with formic
acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying
reagent. It was shown that the replacement of the central A · T by the G · C
pair in the methylation site did not affect enzyme–DNA interaction, whereas the
use of a substrate with one strand methylated (monomethylated substrate)
instead of the unmethylated substrate dramatically changes the DNA contacts.
The binding constants of unmethylated and monomethylated substrates with
methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were
calculated.
II. BIOENERGETICS
AND PHOTOSYNTHESIS
Abdullaev Z.K., Bodrova M.E., Chernyak B.V., Dolgikh D.A., Kluck R.M.,
Perverzev M.O., Arseniev A.S., Efremov R.G., Kirpichnikov M.P., Mokhova E.N.,
Newmeyer D.D., Roder H., Skulachev V.P.
Biochemical Journal 362 (2002) 749-754
A cytochrome c
mutant lacking apoptogenic function but competent in electron transfer and
antioxidant activities has been constructed. To this end, mutant species of
horse and yeast cytochromes c with substitutions in the N-terminal a-helix or
position 72 were obtained. It was found that yeast cytochrome c was much
less effective than the horse protein in activating respiration of rat liver
mitoplasts deficient in endogenous cytochrome c as well as in inhibition
of H2O2 production by the initial segment of the
respiratory chain of intact rat heart mitochondria. The major role in the
difference between the horse and yeast proteins was shown to be played by the
amino acid residue in position 4 (glutamate in horse, and lysine in yeast;
horse protein numbering). A mutant of the yeast cytochrome c containing
K4E and some other 'horse' modifications in the N-terminal a-helix, proved
to be (i) much more active in electron transfer and antioxidant activity than
the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c,
inactive in caspase stimulation, even if added in 400-fold excess compared with
the horse protein. Thus this mutant seems to be a good candidate for knock-in
studies of the role of cytochrome e-mediated apoptosis, in contrast with
the horse K72R. K72G. K72L and K72A mutant cytochromes that at low
concentrations were less active in apoptosis than the wild-type, but were quite
active when the concentrations were increased by a factor of 2-12.
Antonenko Y.N., Borisenko V., Melik-Nubarov N.S., Kotova E.A., Woolley
G.A.
Biophysical Journal 82 (2002) 1308-1318
The effects of
different anionic polymers on the kinetic properties of ionic channels formed
by neutral gramicidin A (gA) and its positively charged analogs
gramicidin-tris(2- aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine
(gram-EDA) in a bilayer lipid membrane were studied using a method of
sensitized photoinactivation. The addition of Konig's polyanion caused
substantial deceleration of the photoinactivation kinetics of gram-TAEA
channels, which expose three positive charges to the aqueous phase at both
sides of the membrane. In contrast, channels formed of gram-EDA, which exposes
one positive charge, and neutral gA channels were insensitive to Konig's
polyanion. The effect strongly depended on the nature of the polyanion added,
namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive,
whereas modified polyacrylic acid induced deceleration of the channel kinetics
at high concentrations. In addition, DNA was able to prevent the action of
Konig's polyanion. In single-channel experiments, the addition of Konig's
polyanion resulted in the appearance of long-lived gram-TAEA channels. The
deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic
interaction of the polyanion with gram-TAEA that reduces the mobility of
gram-TAEA monomers and dinners in the membrane via clustering of
channels.
Azarkina N., Konstantinov A.A.
Journal of Bacteriology 184 (2002) 5339-5347
At a pH of less
than or equal to 7, respiration of Bacillus subtilis cells on endogenous
substrates shut down almost completely upon addition or an uncoupler (carbonyl
cyanide m-chlorophenylhydrazone [CCCP]) and a K+-ionophore
(valinomycin). The same effect was observed with cell spheroplasts lacking the
cell wall. The concentration of CCCP required for 50% inhibition of the
endogenous respiration in the presence of K+- valinomycin was below
100 nM. Either CCCP or valinomycin alone was much less efficient than the
combination of the two. The inhibitory effect was easily reversible and
depended specifically on the H+ and K+ concentrations in
the medium. Similar inhibition was observed with respect to the reduction of
the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and
N,N,N',N'-tetramethyl-p-phenylenediamine cation (TMPD+), which
intercept reducing equivalents at the level of menaquinol. Oxidation of the
reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling.
The same loss of the electron transfer activities as induced by the uncoupling was
observed upon disruption of the cells during isolation of the membranes; the
residual activities were not further inhibited by the uncoupler and ionophores.
We conclude that the menaquinone-dependent electron transfer in the B.
subtilis respiratory chain is facilitated, thermodynamically or
kinetically, by membrane energization. A requirement for an energized state of
the membrane is not a specific feature of succinate oxidation, as proposed in
the literature, since it was also observed in a mutant of B. subtilis
lacking succinate:quinone reductase as well as for substrates other than
succinate. Possible mechanisms of the energy-dependent regulation of
menaquinone- dependent respiration in B. subtilis are discussed.
Bertsova Y.V., Bogachev A.V.
Biochemistry (Moscow) 67 (2002) 22-626
A part of the
gene encoding cbb3-type cytochrome oxidase CcoN Subunit was
cloned from Azotobacter vinelandii and a mutant strain of this bacterium
with disrupted ccoN gene was constructed. In contrast to the wild type strain,
this one is unable to oxidize cytochromes c4 and c5.
Thus, the A. vinelandii respiratory chain is shown to contain cbb3-type
cytochrome c oxidase. It is also shown that the activity of this enzyme
is not necessary for diazotrophic growth of A. vinelandii at high oxygen
concentrations
Bogachev A.V., Bertsova Y.V., Ruuge E.K., Wikstrom M., Verkhovsky M.I.
Biochim Biophys Acta 1556 (2002) 113-120
Two radical
signals with different line widths are seen in the Na+-translocating
NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by
EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is
assigned as a neutral flavosemiquinone. The second radical is observed in the
reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The
time course of Na+-NQR reduction by NADH, as monitored by
stopped-flow optical spectroscopy, shows three distinct phases, the spectra of
which suggest that they correspond to the reduction of three different flavin
species. The first phase is fast both in the presence and absence of sodium,
and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site.
The rates of the other two phases are strongly dependent on sodium
concentration, and these phases are attributed to reduction of two covalently
bound FMN's. Combination of the optical and EPR data suggests that a neutral
FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced
to the fully reduced flavin by NADH. The other FMN moiety is initially
oxidized, and is reduced to the anionic flavosemiquinone. One-electron
transitions of two discrete flavin species are thus assigned as
sodium-dependent steps in the catalytic cycle of Na+-NQR.
Borisov A.Yu., Kuznetsova S.A.
Biochemistry (Moscow) 67 (2002) 1224-1229
A locus for
binding a mobile water molecule was searched for in the immediate vicinity of
the special pair in the reaction center. Using the PROTEUS PC_program (a part
of the GRASP package) atomic structures of the reaction centers were analyzed
in purple bacteria Rhodopseudomonas viridis and Rhodobacter
sphaeroides. In both structures the loci for binding mobile water molecules
were found at the distance of about 4.5 A from the middle of the special pair
in the reaction center. The reorientation of a hydrogen atom of this water
molecule in the electric field of the excited special pair required energy of
no less than 40 MeV that corresponded to predictions of the water_polarization
model of trapping of electron excitation which was developed by M.V. Fok and
one of the authors of this article.
Borisov A.Yu., Vasil’kov S.L.
Biophysica 47 (2002) 272-283
Homogeneous
pigment assemblies were considered, which are close in their characteristics to
those in the purple bacteria Rhodospirillum rubrum and some others.
Expressions were derived for the limit lifetime of electron excitations in these
assemblies and for the limit quantum yield of trapping of excitations by
reaction centers, all of which are in an active state. Mathematical modeling
confirmed that these quantities depend on three parameters of molecular
assemblies: the ratio between the number of core bacteriochlorophyll molecules
and the number of reaction centers, the rate constant for trapping of electron
excitations by reaction centers, and the rate constant for trivial deactivation
of electron excitations in all molecules. It was shown that, with increasing
energy migration rate constants, the lifetime of electron excitations and the
quantum yield of trapping of electron excitations by reaction centers tend to
their respective limit values. The closeness of the experimentally measured
values of these quantities to the said limit values argues in favor of that the
corresponding photosynthetic units are migration-limited and that the energy
transfer from a molecular antenna to reaction centers is highly efficient.
Borisov V.B.
Molecular Aspects of Medicine 23 (2002) 385-412
Here,
relationships between alterations in tissue-specific content, protein
structure, activity, and/or assembly of respiratory complexes III and IV
induced by mutations in corresponding genes and various human pathologies are
reviewed. Cytochrome bc1 complex and cytochrome c
oxidase (COX) deficiencies have been detected in a heterogeneous group of
neuromuscular and non-neuromuscular diseases in childhood and adulthood,
presenting a number of clinical phenotypes of variable severity. Such disorders
can be caused by mutations located either in mitochondrial genes or in nuclear
genes encoding structural subunits of the complexes or corresponding assembly
factors/chaperones. Of the defects in mitochondrial DNA genes, mutations in
cytochrome b subunit of complex III, and in structural subunits I-III of
COX have been described to date. As to defects in nuclear DNA genes, mutations
in genes encoding the complexes assembly factors such as the BCS1L protein for
complex III; and SURF-1, SCO1, SCO2, and COX10 for complex IV have been
identified so far.
Borisov V.B., Liebl U., Rappaport F., Martin J.L., Zhang J., Gennis
R.B., Konstantinov A.A., Vos M.H.
Biochemistry 41 (2002) 1654-1662
Femtosecond
spectroscopy was performed on CO-liganded (fully reduced and mixed-valence
states) and O2-liganded quinol oxidase bd from Escherichia
coli. Substantial polarization effects, unprecedented for optical studies
of heme proteins, were observed in the CO photodissociation spectra, implying
interactions between heme d (the chlorin ligand binding site) and the
close-lying heme b595 on the picosecond time scale; this
general result is fully consistent with previous work [Vos, M. H., Borisov, V.
B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad.
Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and
anisotropic polarization conditions and additional flash photolysis nanosecond
experiments on a mutant of cytochrome bd mostly lacking heme b595
allow to attribute the features in the well-known but unusual CO dissociation
spectrum of cytochrome bd to individual heme d and heme b595
transitions. This renders it possible to compare the spectra of CO dissociation
from reduced and mixed-valence cytochrome bd under static conditions and
on a picosecond time scale in much more detail than previously possible. CO
binding/dissociation from heme d is shown to perturb ferrous heme b595,
causing induction/loss of an absorption band centered at ~435 nm. In addition,
the CO photodissociation-induced absorption changes at 50 ps reveal a
bathochromic shift of ferrous heme b595relative to the static
spectrum. No evidence for transient binding of CO to heme b595
after dissociation from heme d is found in the picosecond time range. The
yield of CO photodissociation from heme d on a time scale of <15 ps
is found to be diminished more than 3-fold when heme b595 is
oxidized rather than reduced. In contrast to other known heme proteins,
molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd
at all, indicating a unique structural and electronic configuration of the
diheme active site in the enzyme.
Domnina L.V., Ivanova O.Y., Cherniak B.V., Skulachev V.P., Vasiliev J.M.
Biochemistry (Moscow) 67 (2002) 37-746
Changes in
cytoskeletal structures have been investigated during apoptosis of epithelial
HeLa cells induced by tumor necrosis factor-a (TNF-a). Shape and surface cell activity
were investigated by time-lapse video microscopy, and changes of the
cytoskeletal structure were studied by immune fluorescent microscopy. Addition
of TNF-a to HeLa cell
culture caused early disruption of the actin cytoskeleton and
vinculin-containing focal contacts, keratin filaments, and microtubules.
Rounding of cells, general blebbing, and nuclear fragmentation were observed at
the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077,
suppressed blebbing (but not cell rounding) and activated the development of
apoptosis. The latter suggests that in contrast to blebbing the general
rounding does not depend on increased contractility of actomyosin cortex. These
cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells
and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding
antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal
structures significantly attenuated antiapoptotic activity of Bcl-2 in the
HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by
cytoskeletal inhibitors may be attributed to the altered distribution of cell
organelles, especially, mitochondria.
Efanov A.M., Appelskog I.B., Abdel-Halim S.M., Khan A., Branstrom R.,
Larsson O., Ostenson C.G., Mest H.J., Berggren P.O., Efendic S., Zaitsev S.V.
American Journal of Physiol. Endocrinol. Metab. 282 (2002) E117-124
The
insulinotropic activity of the imidazoline derivative RX871024 was compared in
pancreatic islets from nondiabetic Wistar rats and spontaneously diabetic
Goto-Kakizaki (GK) rats. RX871024 significantly stimulated insulin secretion in
islets from both animal groups. The insulinotropic activity of RX871024 was
higher than that of the sulfonylurea glibenclamide. This difference was more
pronounced in islets from GK rats compared with Wistar rat islets. More
importantly, RX871024 substantially improved glucose sensitivity in diabetic
beta-cells, whereas glibenclamide stimulated insulin secretion about twofold
over a broad range of glucose concentrations in nondiabetic and diabetic rats.
RX871024 induced a faster increase in cytosolic free Ca2+ concentration
and faster inhibition of ATP-dependent K+ channel activity in GK rat
islets compared with Wistar rat islets. RX871024 also induced a more pronounced
increase in diacylglycerol concentration in GK rat islets. These data support
the idea that imidazoline compounds can form the basis for the development of
novel drugs for treatment of type 2 diabetes, which can restore glucose
sensitivity in diabetic beta-cells.
Efendic S., Efanov A.M., Berggren P.O., Zaitsev S.V.
Diabetes 51 (2002) S448-S454
The imidazoline
RX871024 increased basal- and glucose-stimulated insulin release in vitro and
in vivo. The compound inhibited activity of ATP-sensitive K+
channels as well as voltage-gated K+ channels, which led to membrane
depolarization, an increase in the cytosolic Ca2+ concentration ([Ca2+]i),
and insulin release. Importantly, RX871024 also enhanced the insulinotropic
effect of glucose in cells with clamped [Ca2+]i but in
the presence of high ATP and Ca2+ concentration inside the cell. We
believe that the latter effect on insulin exocytosis was at least in part
mediated by a rise in diacylglycerol, which then activated protein kinase C
(PKC) and increased the generation of arachidonic acid (AA) metabolites.
Activation of both the PKC and AA pathways resulted in potentiation of glucose
effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282
did not block ATP-dependent K+ channels, but similarly to RX871024,
it stimulated insulin secretion in depolarized or permeabilized islets.
Accordingly, BL11282 did not influence glucose and insulin levels under basal
conditions either in vitro or in vivo, but it markedly enhanced the
insulinotropic effects of glucose. BL11282 restored the impaired insulin
response to glucose in islets from spontaneously diabetic GK rats. We conclude
that BL11282 belongs to a new class of insulinotropic compounds that
demonstrate a strong glucose-dependent effect on insulin exocytosis.
Functional analysis of the N+/H+
antiporter encoding genes of the cyanobacterium Synechocystis PCC 6803
Elanskaya I.V., Karandashova I.V., Bogachev A.V., Hagemann M.
Biochemistry (Moscow) 67 (2002) 32-440
The role of
putative Na+/H+ antiporters encoded by nhaS1 (slr-1727),
nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response
and internal pH regulation of the cyanobacterium Synechocystis PCC 6803
was investigated. For this purpose the mutants (single, double, and triple) impaired
in genes coding for Na+/H+ antiporters were constructed
using the method of interposon mutagenesis, PCR analyses of DNA demonstrated
that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and
the mutants contained only inactivated copies of the corresponding genes. Na+/H+
antiporter encoded by nhaS3 was essential for viability of Synechocystis
since no completely segregated mutants were obtained. The steady-state
intracellular sodium concentration and Na+/H+ antiporter
activities were found to be the same in the wild type and all mutants. No
differences were found in the growth rates of wild type and mutants during
their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as
well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes
coding for Na+/H+ antiporters was studied. No induction
of any Na+/H+ antiporter encoding gene expression was
found in wild type or single mutant cells grown under high salt or at different
pH values. Nevertheless, in cells of double and triple mutants adapted to high
salt or alkaline pH some of the remaining Na+/H+
antiporter encoding genes showed induction. These results might indicate that
some of Na+/H+ antiporters can functionally replace each
other under stress conditions in Synechocystis cells lacking the
activity of more than one antiporter.
Feniouk B.A., Cherepanov D.A., Voskoboynikova N.E., Mulkidjanian A.Y.,
Junge W.
Biophysical Journal 82 (2002) 1115-1122
ATP synthase is
a unique rotary machine that uses the transmembrane electrochemical potential
difference of proton D? over tilde +H to synthesize ATP from ADP and inorganic
phosphate. Charge translocation by the enzyme can be most conveniently followed
in chromatophores of phototrophic bacteria (vesicles derived from invaginations
of the cytoplasmic membrane). Excitation of chromatophores by a short flash of
light generates a step of the proton-motive force, and the charge transfer,
which is coupled to ATP synthesis, can be spectrophotometrically monitored by
electrochromic absorption transients of intrinsic carotenoids in the coupling
membrane. We assessed the average number of functional enzyme molecules per
chromatophore vesicle. Kinetic analysis of the electrochromic transients
plus/minus specific ATP synthase inhibitors (efrapeptin and venturicidin)
showed that the extent of the enzyme-related proton transfer dropped as a
function of the inhibitor concentration, whereas the time constant of the
proton transfer changed only marginally. Statistical analysis of the kinetic
data revealed that the average number of proton- conducting F0F1-molecules
per chromatophore was approximately one. Thereby chromatophores of Rhodobacter
capsulatus provide a system where the coupling of proton transfer to ATP
synthesis can be studied in a single enzyme/single vesicle mode.
Feniouk B.A., Cherepanov D.A., Junge W., Mulkidjanian
A.Y.
Biochim. Biophys. Acta 1506 (2001) 189-203
F0F1-ATP
synthase (H+-ATP synthase, F0F1) utilizes the
transmembrane protonmotive force to catalyze the formation of ATP from ADP and
inorganic phosphate (Pi). Structurally the enzyme consists of a
membrane-embedded proton-translocating F0 portion and a protruding
hydrophilic F1 part that catalyzes the synthesis of ATP. In
photosynthetic purple bacteria a single turnover of the photosynthetic reaction
centers (driven by a short saturating flash of light) generates protonmotive
force that is sufficiently large to drive ATP synthesis. Using isolated
chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash
induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in
parallel to the transmembrane charge transfer through F0F1
(by following the decay of electrochromic bandshifts of intrinsic carotenoids).
With the help of specific inhibitors of F1 (efrapeptin) and of F0
(venturicidin), we decomposed the kinetics of the total proton flow through F0F1
into (i) those coupled to the ATP synthesis and (ii) the de-coupled
proton escape through F0. Taking the coupled proton flow, we
calculated the H+/ATP ratio; it was found to be 3.3±0.6 at a large driving force (after
one saturating flash of light) but to increase up to 5.1±0.9 at a smaller driving force
(after a half-saturating flash). From the results obtained, we conclude that
our routine chromatophore preparations contained three subsets of chromatophore
vesicles. Chromatophores with coupled F0F1 dominated in
fresh material. Freezing/thawing or pre-illumination in the absence of ADP and
Pi led to an increase in the fraction of chromatophores with at
least one de-coupled F0(F1). The disclosed fraction of
chromatophores that lacked proton-conducting F0(F1)
(approx. 40% of the total amount) remained constant upon these treatments.
Galperin M.Y., Gaidenko T.A., Mulkidjanian A.Y.,
Nakano M., Price C.W.
FEMS Microbiol Letters 205 (2001) 17-23
MHYT, a new
conserved protein domain with a likely signaling function, is described. This
domain consists of six transmembrane segments, three of which contain conserved
methionine, histidine, and tyrosine residues that are projected to lie near the
outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this
domain forms the N-terminus of the sensor histidine kinase Slr2098. In
Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the
N-terminal part of a three-domain protein together with previously described
GGDEF and EAL domains, both of which have been associated with signal
transduction due to their presence in likely signaling proteins. In Bacillus
subtilis YkoW protein, an additional PAS domain is found between the MHYT
and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth
alterations, consistent with a non-essential, signaling role of this protein. A
model of the membrane topology of the MHYT domain indicates that its conserved
residues could coordinate one or two copper ions, suggesting a role in sensing
oxygen, CO, or NO.
Inositol hexakisphosphate
promotes dynamin mediated endocytosis
Hoy M., Efanov A.M., Bertorello A.M., Zaitsev S.V., Olsen H.L., Bokvist
K., Leibiger B., Leibiger I.B., Zwiller J., Berggren P.O., Gromada J.
Proc. Natl. Acad. Sci. USA 99 (2002) 6773-6777
Membrane
homeostasis is maintained by exocytosis and endocytosis. The molecular
mechanisms regulating the interplay between these two processes are not clear.
Inositol hexakisphosphate (InsP6) is under metabolic control and
serves as a signal in the pancreatic beta cell stimulus-secretion coupling by
increasing Ca2+- channel activity and insulin exocytosis. We now
show that InsP6 also promotes dynamin I-mediated endocytosis in the pancreatic
beta cell. This effect of InsP6 depends on calcineurin-induced
dephosphorylation and is accounted for by both activation of protein kinase C
and inhibition of the phosphoinositide phosphatase synaptojanin and thereby
formation of phosphaticlylinositol 4,5-bisphosphate. In regulating both
exocytosis and endocytosis, InsP6 thus may have an essential
integral role in membrane trafficking.
Isaev N.K., Stelmashook E.V., Dirnagl U., Andreeva N.A., Manuhova L.,
Vorobjev V.S., Sharonova I.N., Skrebitsky V.G., Victorov I.V., Katchanov J.,
Weih M., Zorov D.B.
Neuroscience 113 (2002) 47-53
Pretreatment
with 10 ?M of the antifungal drug
clotrimazole potently reduced the death of cultured rat cerebellar granule
cells induced by oxygen/glucose deprivation, and the excitotoxic effect of
glutamate on cultured hippocampal neurons and cerebellar granule cells. In
patch-clamped hippocampal pyramidal neurons, 10-50 ?M clotrimazole caused a decrease in the amplitude of
N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced
intracellular Ca2+ overload, as measured by Fluo-3 confocal
fluorescence imaging, while clotrimazole reduced Ca2+ overload and
promoted the recovery of intracellular calcium homeostasis after glutamate
treatment Using tetra-methylrhodamine ethyl ester fluorescence as a marker of
mitochondrial membrane potential we found that clotrimazole prevented the
glutamate-induced loss of mitochondrial membrane potential. Our data provide
evidence that the protective effect of clotrimazole against oxygen/glucose
deprivation and excitotoxicity is due to the ability of this drug to partially
block NMDA receptor-gated channel, thus causing both reduced calcium overload
and lower probability of the mitochondrial potential collapse.
Klishin S.S., Junge W., Mulkidjanian A.Y.
Biochim. Biophys. Acta 1553 (2002) 177-182
The effect of
Zn2+ on the rates of electron transfer and of voltage generation in
the cytochrome bc1 (complex bc1) was
investigated under excitation of Rhodobacter capsulatus chromatophores
with flashing light. When added, Zn2+ retarded the oxidation of
cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its
high potential heme bh (in the absence of Zn2+
this reaction vas masked by the fast re- oxidation of the heme). The effect was
accompanied by the deceleration of both the cytochrome c1
reduction (as monitored at 552-570 nm) and the generation of transmembrane
voltage (monitored by electrochromism at 522 nm). At Zn2+ <
100 ?M the reduction of heme bh
remained 10 times faster than other reactions. The kinetic discrepancy was
observed even after an attenuated flash, when bc1 turned over
only once. These observations (1) raise doubt on the notion that the
transmembrane electron transfer towards heme bh is the main
electrogenic reaction in the cytochrome bc1 complex, (2)
imply an allosteric link between the site of heme bh
oxidation and the site of cytochrome c1 reduction at the
opposite side of the membrane, and (3) indicate that the internal
redistribution of protons might account for the voltage generation by the
cytochrome bc1 complex.
Krikunova M., Kummrow A., Voigt B., Rini M., Lokstein H., Moskalenko A.,
Scheer H., Razjivin A., Leupold D.
FEBS Letters 528 (2002) 227-229
Native and
carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2)
were investigated by simultaneous two-photon excited (between 1300-1500 nm)
fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in
both samples. The spectral position of the 2Ag-
state of rhodopin is indicated by a diminuition of the bacteriochlorophyll TPF
in native LH2. In conclusion, comparison to carotenoid-depleted samples is a
conditio sine qua non for unambiguous interpretation of similar experiments.
Kinetically
different populations of O-pyromellityl-gramicidin channels induced by
poly-L-lysines in lipid bilayers
Krylov A.V., Rokitskaya T.I., Kotova E.A., Yaroslavov A.A., Antonenko
Y.N.
Journal of Membrane Biology 189 (2002) 119-130
Clustering of
membrane proteins, in particular of ion channels, plays an important role in
their functioning. To further elucidate the mechanism of such ion channel
activity regulation, we performed experiments with a model system comprising
the negatively-charged gramicidin analog, O- pyromellitylgramicidin (OPg) that
forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect
of polylysines on the kinetics of OPg channels in BLM was studied by the method
of sensitized photoinactivation. As found in our previous work, the interaction
of polylysine with OPg led to the deceleration of the OPg photoinactivation
kinetics, i.e., to the increase in the characteristic time of OPg
photoinactivation. It was shown here that in a certain range of polylysine concentrations
the photoinactivation kinetics displayed systematic deviations from a
monoexponential curve and was well described by a sum of two exponentials. The
deviations from the monoexponential approximation were more pronounced with
polylysines having a lower degree of polymerization. These deviations increased
also upon the elevation of the ionic strength of the bathing solution and the
addition of calcium ions. A theoretical model is presented that relates the OPg
photoinactivation kinetics at different concentration ratios of OPg and
polylysine to the distribution of OPg molecules among OPg-polylysine clusters
of different stoichiometry. This model is shown to explain qualitatively the
experimental results, although the quantitative description of the whole body
of evidence requires further development, assuming that the interaction of
polylysine with OPg causes segregation of membrane domains enriched in OPg
channels. The single-channel data, which revealed the insensitivity of the
single-channel lifetime of OPg to the addition of polylysine, are in good
agreement with the theoretical model.
Lee A., Kirichenko A., Vygodina T., Siletsky S.A., Das T.K., Rousseau
D.L., Gennis R., Konstantinov A.A.
Biochemistry 41 (2002) 8886-8898
Cytochrome c
oxidase (COX) from R. sphaeroides contains one Ca2+ ion per
enzyme that is not removed by dialysis versus EGTA. This is similar to COX from
Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A.,
Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga,
A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in
contrast to the bovine oxidase, which binds Ca2+ reversibly. A series
of R. sphaeroides mutants with replacements of the E54, Q61, and D485
residues, which form the Ca2+ coordination sphere in subunit 1, has
been generated. The substitutions for the E54 residue do not assemble normally.
Mutants with the Q61 replacements are active and retain the tightly bound Ca2+;
their spectra are not perturbed by added Call or EGTA. The D485A mutant is
active, binds to Ca2+ reversibly, like the mitochondrial oxidase,
and exhibits the red shift in the heme a absorption spectrum upon Ca2+
binding for both reduced and oxidized states of heme a. The K-d
value of 6 nM determined by equilibrium titrations is much lower than that
reported for the homologous D477A mutant of Paracoccus denitrificans or
for bovine COX (K-d = 1-3 ?M). The rate of Ca2+ binding with the D485A oxidase (kon
= 5 x 103 M-1 s-1) is comparable to that
observed earlier for bovine COX, but the off-rate is extremely slow (similar
to10-3 s-1) and highly temperature-dependent. The koff/kon
ratio (190 nM) is about 30-fold higher than the equilibrium K-d of 6 nM,
indicating, that formation of the Ca2+-adduct may involve more than
one step. Sodium ions reverse the Ca2+-induced red shift of heme a
and dramatically decrease the rate of Ca2+ binding to the D485A
mutant COX. With the D485A mutant, 1 Ca2+ competes with 1 Na+
for the binding site, whereas 2 Na+compete with 1 Ca2+
for binding to the bovine oxidase. This finding indicates that the aspartic
residue D442 (a homologue of R. sphaeroides D485) may be the second Na+binding
site in bovine COX. No effect of Ca2+ binding to the D485A mutant is
evident on either the steady-state enzymatic activity or several time-resolved
partial steps of the catalytic cycle. It is proposed that the tightly bound
Call plays a structural role in the bacterial oxidases while the reversible
binding with the mammalian enzyme may be involved in the regulation of
mitochondrial function.
Maltseva E.A., Antonenko Y.N., Melik-Nubarov N.S., Yaguzhinsky L.S.
Biologicheskie Membrany 19 (2002) 347-350
The effects of
synthetic amphipathic polyanions (Konig polyanion and a copolymer of
polyacrylic acid with sterylacrylate) on the carrier-mediated ion transport
across planar bilayer lipid membranes (BLM) have been studied. The binding of
these polymers to the membrane increased the cationic current (complex of K+-valinomycin)
and decreased the anionic current (TTFB-) across the membrane. Hydrophylic polyanions
lacking large hydrophobic fragments did not affect the ionic currents across
the BLM. It is concluded that the effect of polyanions on the carrier-induced
ionic transport is mediated by the binding of hydrophobic fragments of
polyanions to the BLM. This leads to the change in the BLM cur-rent due to the
appearance of negative surface charge. The mechanism of the action of Konig
polyanion on the transport systems of mitochondria is discussed.
Mildaziene V., Nauciene Z., Baniene R., Demin O., Krab K.
Molecular Biology Reports 29 (2002) 35-40
Modular kinetic
analysis reveals that the environmental pollutant 2,2',5,5'-tetrachlorobiphenyl
(2,2',5,5'-TCB) affects a large number of steps in oxidative phosphorylation in
rat liver mitochondria. 2,2',5,5'-TCB increases membrane permeability to ions,
and inhibits NADH dehydrogenase, cytochrome bc1, cytochrome
oxidase (all in the respiratory chain) and ATP-synthase (in the phosphorylation
subsystem). Surprisingly, flux control distribution does not change. A kinetic
model for oxidative phosphorylation was used to simulate these findings, and it
was found that combined large changes in the processes indicated indeed left
the flux control largely unchanged. In addition, computational analysis with
the model indicated that the adenine nucleotide translocator might be inhibited
by 2,2',5,5'-TCB.
Moehren G., Markevich N., Demin O., Kiyatkin A., Goryanin I., Hoek J.B.,
Kholodenko B.N.
Biochemistry 41 (2002) 306-320
Stimulation of
isolated hepatocytes with epidermal growth factor (EGF) causes rapid tyrosine
phosphorylation of the EGF receptor (EGFR) and adapter/target proteins, which
was monitored with 1 and 2 s resolution at 37, 20, and 4 degrees C. The
temporal responses detected for multiple signaling proteins involve both
transient and sustained phosphorylation patterns, which change dramatically at
low temperatures. To account quantitatively for complex responses, we employed
a mechanistic kinetic model of the EGFR pathway, formulated in molecular terms
as cascades of protein interactions and phosphorylation and dephosphorylation
reactions. Assuming differential temperature dependencies for different
reaction groups, such as SH2 and PTB domain-mediated interactions, the EGFR
kinase, and the phosphatases, good quantitative agreement was obtained between
computer-simulated and measured responses. The kinetic model demonstrates that,
for each protein-protein interaction, the dissociation rate constant, koff,
strongly decreases at low temperatures, whereas this decline may or may not be
accompanied by a large decrease in the kon value.
Temperature-induced changes in the maximal activities of the reactions
catalyzed by the EGFR kinase were moderate, compared to such changes in the Vmax
of the phosphatases. However, strong changes in both the Vmax and Km
for phosphatases resulted in moderate changes in the Vmax/Km
ratio, comparable to the corresponding changes in EGFR kinase activity, with a
single exception for the receptor phosphatase at 4 degrees C. The model
suggests a significant decrease in the rates of the EGF receptor dimerization
and its dephosphorylation at 4 degrees C, which can be related to the phase
transition in the membrane lipids. A combination of high-resolution
experimental monitoring and molecular level kinetic modeling made it possible
to quantitatively account for the temperature dependence of the integrative
signaling responses.
Muntyan M.S, Dinarieva T.Yu., Baev M.V., Netrusov A.I.
Archives of Biochemistry and Biophysics 398 (2002) 118-124
Membranes of
the obligate methylotroph Methylobacillus flagellatus KT contained hemes
B, O, and C and cytochromes b, o, and c both in batch
and in continuous cultures. Neither heme A nor heme D was
detected in the membranes. The cytochromes o and bb were the main
components reversibly binding carbon monoxide (CO) in the terminal part of the
respiratory chain. The a-region and especially the a-peaks at 568 and 573 nm and
the a-troughs at 586
and 592 on the CO-difference spectra were diagnostic for the cytochromes o
and bb, respectively. The cytochrome o content increased up to
1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h-1
under methanol limitation. By contrast, the level of the CO-binding cytochrome bb
was not affected by methanol concentration but its content increased up to 1.9
times when the level of oxygen decreased from 95 to 21 mM under the constant dilution rate (m = 0.55 h-1)). The
maximum ratio between the cytochromes o and bb reached 2 during
continuous cultivation under methanol-limited conditions (m = 0.55 h-1)), whereas
the minimum ratio between them was about 0.7 during batch cultivation at
stationary phase of growth. The synthesis of the CO-binding cytochrome bb
but not of the cytochrome o in M. flagellatus KT was assumed to
depend on the ambient redox potential of the medium. The cytochrome o
synthesis was supposed to depend on the transmembrane gradient of protons (d-mH+).
Muntyan M.S., Tourova T.P., Lysenko A.M., Kolganova T.V., Fritze D.,
Skulachev V.P.
Extremophiles 6 (2002) 195-199
The systematic
position of the alkaliphilic and halotolerant strain Bacillus sp. FTU
was refined in view of the comprehensive taxonomic revision of the group of
alkaliphilic and alkalitolerant Bacillus strains. Sequence analysis of
almost the entire 16S rRNA gene of Bacillus sp. FTU revealed 99.8%
homology with two Bacillus pseudofirmus strains. Subsequent DNA-DNA
hybridization analysis confirmed the close relationship of Bacillus sp.
FTU with the type strain of B. pseudofirmus (the level of homology reached
86%). Results of physiological and biochemical characterizations relevant for
the group clearly underlined the positioning of strain FTU within this species.
It is therefore concluded that Bacillus sp. FTU represents a strain of
the alkaliphilic species B. pseudofirmus and is to be renamed as B.
pseudofirmus FTU. The phylogeny of different Bacillus species is
discussed using N- terminal sequence homologies of some caa(3)-type oxidase
subunits.
Exciton-vibrational
relaxation and transient absorption dynamics in LH1 of Rhodopseudomonas
viridis: A Redfield theory approach
Novoderezhkin V., van Grondelle R.
Journal of Physical Chemistry B 106 (2002) 6025-6037
We explain the
ultrafast transient absorption (TA) dynamics in the core LH1 antenna of Rhodopseudomonas
viridis at 77 K (Monshouwer, R. Baltuska. A.: van Mourik, F.; van
Grondelle, R. J, Phys. Chem. A 1998, 102, 4360) using a disordered exciton
model with strong coupling to two vibrational modes and weak coupling of
vibrational and electronic coordinates to the thermal bath. A quantitative fit
of the excitation wavelength- dependent TA dynamics was obtained using the
density matrix equation with the Redfield relaxation operator. The sequential
and coherent contributions to the TA dynamics were analyzed separately. The
time-dependent red shift of TA was explained in terms of exciton relaxation.
The lifetimes of higher exciton states of 12-75 fs (depending on the state)
were determined from the fit.
Palamarchuk L.A., Mansurova S.E., Starkov A.A.
Biochemistry (Moscow) 67 (2002) 468-472
Earlier we
reported that some thyroid and steroid hormones and also 6-ketocholestanol used
in micromolar concentrations modulated the effects of protonophoric uncouplers
on isolated mitochondria (Starkov et al, (1997) Biochim. Biophys. Acta, 1318,
173-183). In the present study we investigated the effects of a thyroid
hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and
mitochondria isolated from these cells. The resting (oligomycin-inhibited)
respiration of the isolated intact lymphocytes was stimulated by the addition
of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of
micromolar concentrations of thyroxin decreased the rate of
uncoupler-stimulated respiration and partially reversed uncoupler-induced
decrease of membrane potential (DY). In
experiments with mitochondria isolated from thymus lymphocytes the re-coupling
effect of thyroxine was not observed. In this case thyroxine did not influence
mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation
of respiration and DY decrease
induced with another uncoupler, SF6847. The data are discussed in terms of a
hypothesis that aromatic uncouplers are transported into the cell by the
thyroxine carrier of the plasma membrane.
Popov V.N., Markova O.V., Mokhova E.N., Skulachev V.P.
Biochim. Biophys. Acta 1553 (2002) 232-237
Effects of cold
exposure in vivo and treatment with laurate, carboxyatractylate,
atractylate. nucleotides, and BSA in vitro on potato tuber mitochondria
ha e been studied. Cold exposure of tubers Cor 48-96 h resulted in some
uncoupling that could be reversed completely by BSA and partially by ADP, ATP,
UDR carboxyatractylate, and atractylate. UDP was less effective than ADP and
ATP. and atractylate was less effective than carboxyatractylate. The recoupling
effects of nucleotides ere absent when the nucleotides were added after
carboxyatractlate. GDP, UDP, and CDP did not recouple mitochondria from either
the control or the cold-exposed tubers. This indicates that the cold-induced
fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to
the operation of the ATP/ADP antiporter, As to the plant uncoupling protein,
its contribution to the uncoupling in tuber is negligible or, under the
conditions used, somehow desensitized to nucleotides.
Rokitskaya T.I., Kotova E.A., Antonenko Yu.N.
Biophysical Journal 82 (2002) 865-873
The effect of
membrane dipole potential on gramicidin channel activity in bilayer lipid
membranes (BLMs) was studied. Remarkably, it appeared that proton conductance
of gramicidin A (gA) channels responded to modulation of the dipole potential
oppositely as compared with gA alkali metal cation conductance. In particular,
the addition of phloretin, known to reduce the membrane dipole potential,
resulted in a decrease in gA proton conductance, on one hand, and an increase
in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol,
the agent raising the membrane dipole potential, provoked an increase in gA
proton conductance as opposed to a decrease in the alkali metal cation
conductance. The peculiarity of the 6-ketocholestanol effect consisted in its
dependence on the H+ concentration. The experiments with the
impermeant dipolar compound, phloridzin, showed that the response of proton
transport through gramicidin channels to varying the membrane dipole potential
did not change qualitatively if the dipole potential of only one monolayer or
both monolayers of the BLM was altered. In contrast to gA proton conductance,
the single-channel lifetime changed similarly with varying the membrane dipole
potential, regardless of the kind of permeant cations (protons or potassium
ions). The results of this study could be tentatively accounted for by an
assumption that one of the rate-limiting steps of proton conduction through
gramicidin channels represents, in fact, movement of negatively charged species
(negative ionic defects) across a membrane.
Rosen G.M., Tsai P., Weaver J., Porasuphatana S., Roman L.J., Starkov
A.A., Fiskum G., Pou S.
Journal of Biological Chemistry 277 (2002) 40275-40280
Tetrahydrobiopterin
(H4B) is a critical element in the nitric-oxide synthase (NOS)
metabolism of l-arginine to l-citrulline and NO.. It has been
hypothesized that in the absence of or under nonsaturating levels of L-arginine
where O2 reduction is the primary outcome of NOS activation, H4B
promotes the generation of H2O2 at the expense of O2-..
The experiments were designed to test this hypothesis. To test this theory, two
different enzyme preparations, H4B-bound NOS I and H4B-free
NOS I, were used. Initial rates of NADPH turnover and O2 utilization
were found to be considerably greater in the H4B-bound NOS I
preparation than in the H4B-free NOS I preparation. In contrast, the
initial generation of O2-. from the H4B-free
NOS I preparation was found to be substantially greater than that measured
using the H4B-bound NOS I preparation. Finally, by spin trapping
nearly all of the NOS I produced O2-., we found that the
initial rate of H2O2 production by H4B-bound
NOS I was considerably greater than that for H4B-free NOS I.
Ruscher K., Freyer D., Karsch M., Isaev N., Megow D., Sawitzki B.,
Priller J., Dirnagl U., Meisel A.
Journal of Neuroscience 22 (2002) 10291-10301
In an in vitro
model of cerebral ischemia (oxygen glucose deprivation, OGD) we investigated
whether erythropoietin (EPO) plays a critical role in ischemic preconditioning.
We found that EPO time and dose-dependently induced protection against OGD in
rat primary cortical neurons. Protection was significant at 5 min and reached a
maximum at 48 hr after EPO application. Protection was blocked by the
coapplication of a soluble Epo receptor (sEpoR) or an antibody against EpoR
(anti-EpoR). Medium transfer from OGD-treated astrocytes to untreated neurons
induced protection against OGD in neurons, which was attenuated strongly by the
application of sEpoR and anti-EpoR. In contrast, medium transfer from
OGD-treated neurons to untreated neurons induced protection against OGD that
did not involve EPO. In astrocytes the OGD enhanced the nuclear translocation
of hypoxia-inducible factor 1 (HIF-1), the major transcription factor
regulating EPO expression. Consequently, transcription of EPO-mRNA was
increased in astrocytes after OGD. Cultured neurons express EpoR, and the Janus
kinase-2 (JAK-2) inhibitor AG490 abolished EPO-induced tolerance against OGD.
Furthermore, EPO-induced neuroprotection as well as phosphorylation of the
proapoptotic Bcl family member Bad was reduced by the phosphoinositide-3 kinase
(PI3K) inhibitor LY294002. The results suggest that astrocytes challenged with
OGD provide paracrine protective signals to neurons. We provide evidence for
the following signaling cascade: HIF-1 is activated rapidly by hypoxia in
astrocytes. After HIF-1 activation the astrocytes express and release EPO. EPO
activates the neuronal EPO receptor and, subsequently, JAK-2 and thereby PI3K.
PI3K deactivates BAD via Akt-mediated phosphorylation and thus may
inhibit hypoxia-induced apoptosis in neurons. Our results establish EPO as an
important paracrine neuroprotective mediator of ischemic preconditioning.
Saprunova V.B., Kazimirchuk S.A., Tonshin A.A., Bakeeva L.E., Yagujinsky
L.S.
Biochemistry (Moscow) 67 (2002) 246-253
The effect of
anoxic incubation of small slices of isolated rat hearts on respiration,
internucleosomal DNA fragmentation, and mitochondrial ultrastructure was
investigated. Anoxic incubation for 72 h induced apoptosis accompanied by
internucleosomal DNA fragmentation and changes in respiration and mitochondrial
ultrastructure. The mitochondrial population was characterized by morphological
heterogeneity. In a significant part of the mitochondrial population there were
signs of mitochondrial swelling and appearance of electron- dense mitochondria.
Anoxia also induced the appearance of an atypical (and previously unknown)
population of small electron- dense mitochondria. They were characterized by
unusual localization inside electron-light mitochondria. Under anoxic
conditions the inner mitochondrial membrane formed electron- dense ordered
structures. All changes described here reflect two opposing processes occurring
in mitochondria: apoptotic destruction and compensatory processes responsible
for maintenance of mitochondria.
Severina I.I., Muntyan M.S., Lewis K., Skulachev V.P.
IUBMB Life 52 (2001) 321-324
Some organic
cations are known to be electrophoretically imported into bacterial cells and
actively extruded from these cells by multidrug resistance (MDR) pumps. We have
studied penetration of plant antimicrobial agents berberine and palmatine and
synthetic antiseptic benzalkonium chloride through black planar phospholipid
membrane (BLM) and membrane of Staphylococcus aureus cells. Gradients of
these cations across BLM generated an electric potential difference.
Penetrating anion tetraphenyl borate and phloretin (a plant substance
decreasing membrane dipole potential) stimulated this effect. Under optimal
conditions, the magnitude of the electric potential was close to theoretical, that
is, 60 mV/10-fold cation gradient. Berberine accumulated in S. aureus
cells as shown by direct measurement of berberine with a berberine-sensitive
electrode. The berberine accumulation was prevented by protonophore CCCP and
was stimulated by mutation in the MDR pump NorA. It is concluded that the plant
alkaloids and benzalkonium are penetrating cations and substrates of an MDR
pump.
Shchepina L.A., Pletjushkina O.Y., Avetisyan A.V., Bakeeva L.E.,
Fetisova E.K., Izyumov D.S., Saprunova V.B., Vyssokikh M.Y., Chernyak B.V.,
Skulachev V.P.
Oncogene 21 (2002) 8149-8157
The release of
cytochrome c from the intermembrane space of mitochondria into the
cytosol is one of the critical events in apoptotic cell death. In the present
study, it is shown that release of cytochrome c and apoptosis induced by
tumor necrosis factor a (TNF) in HeLa cells can be inhibited by (i) overexpression of an
oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial
permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+-ATP-synthase.
Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin
A and oligomycin. The effect of oligomycin is not due to changes in
mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis
since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to
abolish the protective action of oligomycin and (b) aurovertin B (another
inhibitor of H+-ATP-synthase, affecting its F1 component)
do not affect apoptosis. A role of oligomycin-sensitive F0 component
of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is
suggested.
Sjoholm A., Lehtihet M., Efanov A.M., Zaitsev S.V., Berggren P.O.,
Honkanen R.E.
Endocrinology 143 (2002) 4592-4598
In human type 2
diabetes mellitus, loss of glucose-sensitive insulin secretion is an early
pathogenetic event. Glucose is the cardinal physiological stimulator of insulin
secretion from the pancreatic beta-cell, but the mechanisms involved in glucose
sensing are not fully understood. Specific ser/thr protein phosphatase (PPase)
inactivation by okadaic acid promotes Ca2+ entry and insulin
exocytosis in the beta-cell. We now show that glycolytic and Krebs cycle
intermediates, whose concentrations increase upon glucose stimulation, not only
dose dependently inhibit ser/thr PPase enzymatic activities, but also directly
promote insulin exocytosis from permeabilized beta-cells. Thus,
fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate,
and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis,
nonadditive to that of okadaic acid, at micromolar Ca2+
concentrations. In contrast, the effect of GTP is potentiated by okadaic acid,
suggesting that the action of GTP does not require PPase inactivation. We
conclude that specific glucose metabolites and GTP inhibit beta-cell PPase
activities and directly stimulate Ca2+-independent insulin
exocytosis. The glucose metabolites, but not GTP, seem to require PPase
inactivation for their stimulatory effect on exocytosis. Thus, an increase in
phosphorylation state, through inhibition of protein dephosphorylation by
metabolic intermediates, may be a novel regulatory mechanism linking glucose
sensing to insulin exocytosis in the beta-cell.
Skulachev V.P.
Ann. N. Y. Acad. Sci. 959 (2002) 214-237
Programmed
death phenomena appear to be inherent not only in living cells (apoptosis), but
also in subcellular organelles (e.g., self-elimination of mitochondria, called
mitoptosis), organs (organoptosis), and even whole organisms (phenoptosis). In
all these cases, the "Samurai law of biology"--it is better to die
than to be wrong--seems to be operative. The operation of this law helps
complicated living systems avoid the risk of ruin when a system of lower
hierarchic position makes a significant mistake. Thus, mitoptosis purifies a
cell from damaged and hence unwanted mitochondria; apoptosis purifies a tissue
from unwanted cells; and phenoptosis purifies a community from unwanted
individuals. Defense against reactive oxygen species (ROS) is probably one of
the primary evolutionary functions of programmed death mechanisms. So far, it
seems that ROS play a key role in the mito-, apo-, organo-, and phenoptoses,
which is consistent with Harman's theory of aging. Here a concept is described
that tries to unite Weismann's hypothesis of aging as an adaptive programmed
death mechanism and the generally accepted alternative point of view that
considers aging as an inevitable result of accumulation in an organism of
occasional injuries. It is suggested that injury accumulation is monitored by a
system(s) actuating a phenoptotic death program when the number of injuries
reaches some critical level. The system(s) in question are organized in such a
way that the lethal case appears to be a result of phenoptosis long before the
occasional injuries make impossible the functioning of the organism. It is
stressed that for humans these cruel regulations look like an atavism that, if overcome,
might dramatically prolong the human life span.
Programmed death in yeast as
adaptation?
Skulachev V.P.
FEBS Letters 528 (2002) 23-26
During recent
years, several pieces of indirect evidence of a programmed death in yeast have
been published. Among them there are observations that some mammalian pro- or
anti-apoptotic proteins induce or prevent the death of yeast; some toxic
compounds kill yeast at lower concentrations if protein synthesis is operative;
this death, as well as the death due to certain mutations, shows some apoptotic
markers. In April 2002, the yeast programmed death concept received direct
support. Madeo et al. [Madeo et al., Mol. Cell 9 (2002) 911-917] disclosed a
caspase which is activated by H2O2 or aging and is
required for the protein-synthesis-dependent death of yeast. Thus, a specific
apoptosis-mediating protein was identified for the first time in Saccharomyces
cerevisiae. Independently, Severin and Hyman [Severin, F.F., Hyman, A.A.,
Curr. Biol. 12 (2002) R233-R235] discovered that death of yeast, induced by a
high level of a pheromone, is programmed. In particular, the death was found to
be prevented by cycloheximide and cyclosporin A. It required mitochondrial DNA,
cytochrome c and the pheromone-initiated protein kinase cascade. When haploids
of opposite mating types were mixed, some cells died, the inhibitory pattern
being the same as in the case of the killing by pheromone. Inhibition of mating
proved to be favorable for death. Thus, pheromone not only activates mating but
also eliminates yeast cells failing to mate. Such an effect should (i)
stimulate switch of the yeast population from vegetative to sexual
reproduction, and (ii) shorten the life span and, hence, accelerate changing of
generations. As a result, the probability of appearance of new traits could be
enhanced when ambient conditions turned for the worse.
Starkov A.A., Polster B.M., Fiskum G.
Journal of Neurochemistry 83 (2002) 220-228
Abnormal
accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as
Bax, is believed to stimulate mitochondrial generation of reactive oxygen
species (ROS) and contribute to neural cell death during acute ischemic and
traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's
disease. However, the mechanism by which Ca2+ or apoptotic proteins
stimulate mitochondrial ROS production is unclear. We used a sensitive
fluorescent probe to compare the effects of Ca2+ on H2O2
emission by isolated rat brain mitochondria in the presence of physiological
concentrations of ATP and Mg2+ and different respiratory substrates.
In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2
generation and reduced the membrane potential of mitochondria oxidizing
succinate, or glutamate plus malate. In the presence of the respiratory chain
Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2
production by mitochondria oxidizing succinate, and this stimulation was
associated with release of mitochondrial cytochrome c. In the
presence of glutamate plus malate, or succinate, cytochrome c release
and H2O2 formation were stimulated by human recombinant
full-length Bax in the presence of a BH3 cell death domain peptide. These
results indicate that in the presence of ATP and Mg2+, Ca2+
accumulation either inhibits or stimulates mitochondrial H2O2
production, depending on the respiratory substrate and the effect of Ca2+
on the mitochondrial membrane potential. Bax plus a BH3 domain peptide
stimulate H2O2 production by brain mitochondria due to
release of cytochrome c and this stimulation is insensitive to changes
in membrane potential.
Starkov A.A., Wallace K.B.
Toxicol. Sci. 66 (2002) 244-252
Perfluorooctanoate
(PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome
proliferation and interfere with mitochondrial metabolic pathways. Direct measurements
revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA)
uncouple mitochondrial respiration by increasing proton conductance. The
purpose of this investigation was to characterize structural determinants
responsible for the mitochondrial uncoupling effect of several structurally
related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the
N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA),
N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol
[2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic
acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each
test compound was dissolved in ethanol and added directly to an incubation
medium containing substrate-energized rat liver mitochondria. Mitochondrial
respiration and membrane potential were measured concurrently using an oxygen
electrode and a TPP+ -selective electrode, respectively. All of the compounds
tested, at sufficiently high concentrations, had the capacity to interfere with
mitochondrial respiration, albeit via different mechanisms and with
varying potencies. At sufficiently high concentrations, the free acids PFOA and
PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial
inner membrane, which resembled a surfactant-like change in membrane fluidity.
Similar effects were observed with the sulfonamide N-EtFOSE. Another fully
substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of
respiration, the release of cytochrome c, and high-amplitude swelling of
mitochondria. The swelling was prevented by cyclosporin A or by EGTA,
indicating that this compound induced the mitochondrial permeability
transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and
FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of
protonophoric uncouplers. Among these compounds, FOSA was a very potent
uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM.
These data suggest that the protonated nitrogen atom with a favorable pKa is
essential for the uncoupling action of perfluorooctane sulfonamides in
mitochondria, which may be critical to the mechanism by which these compounds
interfere with mitochondrial metabolism to induce peroxisome proliferation in
vivo.
Vyssokikh M.Y., Zorova L., Zorov D., Heimlich G., Jurgensmeier J.M.,
Brdiczka D.
Molecular Biology Reports 29 (2002) 93-96
The mechanism
by which external Bax releases cytochrome c is still controversial and
may also depend on the type of mitochondria and the actual localisation of
cytochrome c. Outer membrane porin acquires high binding affinity for
hexokinase by interacting with the adenine nucleotide translocator (ANT) in the
contact sites. (I) The hexokinase protein was thus used as a tool to isolate
the contact site forming complex between outer membrane porin and inner
membrane ANT from a Triton-X100 extract of brain membranes. (II) A significant
amount of cytochrome c was co-purified with the isolated hexokinase
porin ANT complexes that were reconstituted in phospholipid vesicles.
Bax-DeltaC released the endogenous cytochrome c from the vesicles
without forming unspecific pores. This was shown by loading the vesicles with
malate that was not liberated by Bax- AC. (III) The Bax-DeltaC effect was
dependent on a specific association of cytochrome c with the porin ANT
complex, as dissociation of the complex by bongkrekate abolished the Bax
dependent cytochrome c liberation. (IV) The Bax-AC effect was as well
suppressed by hexokinase phosphorylating glucose.
Wendling M., Lapouge K., van Mourik F., Novoderezhkin V., Robert B., van
Grondelle R.
Chemical Physics 275 (2002) 31-45
By reversible
dissociation of the light-harvesting complex 1 (LH1) of Rhodospirillum
rubrum it is possible to (partly) exchange the bacteriochlorophyll (BChl) a
by zinc- bacteriopheophytin (Zn-BPheo). After reassociation of the protein a
complex is formed which can have different percentages of Zn-BPheo bound to the
polypeptides [Biochemistry 39 (2000) 1091]. Low-temperature absorption spectra
show a shift of the absorption maximum from 886 to 863 nm, when the native LH1
complex is compared to a modified complex containing 90% Zn-BPheo, whereas the
positions of the absorption maxima of BChl a and Zn-BPheo differ by only 6 nm,
when the pigments are bound to the isolated polypeptides. Using an exciton
model with static disorder of site energies based on the ring-like structure of
LH1 we can describe this shift by assuming a difference in the excitonic
coupling originating solely from the different dipole strength of the exchanged
Zn-BPheo compared with the original BChl a. We estimate that in LH1 the
nearest-neighbour interaction energy of two BChl a molecules is around 400 cm-1
and the diagonal disorder is around 600 cm-1. Furthermore, we
determined if the energy transfer in pigment- modified complexes is similar to
native LH1. This can be observed by selectively exciting electronic states
depending on their energy and measuring the polarized emission spectra at low
temperature. The native complex was compared to a complex containing 70%
Zn-BPheo. The fluorescence anisotropy and the shift of the emission maximum in
native LH1 and 70%-Zn-BPheo- LH1 depending on the excitation wavelength can
generally be described within the same disordered exciton model, extended in a
simple way with line shapes. The model proved to be simple and robust when
applied to these engineered light-harvesting complexes.
Yakovlev A.G., Shkuropatov A.Y., Shuvalov V.A.
Biochemistry 41 (2002) 14019-14027
In Rhodobacter
sphaeroides R-26 reaction centers (RCs) the nuclear wave packet induced by
25 fs excitation at 90 K moves on the primary electron donor P* potential
energy hypersurface with initial frequency at approximately 130 cm-1
(monitored by stimulated emission measurement). At the long-wavelength side of
P* stimulated emission at 935 nm the wave packet is transferred to the surface
with P+BA- character at 120, 380, 1.2 fs, etc.
delays (monitored by measurement of the primary electron acceptor P+BA-
band at 1020 nm). However, only beginning from 380 fs delay and later the
relative stabilization of the state P+BA- is
observed. This is accompanied by the electron transfer to bacteriopheophytin HA
(monitored by HA band measurement at 760 nm). The most active mode
of 32 cm-1 in the electron transfer and its overtones up to the
seventh were found in the Fourier transform spectrum of the oscillatory part of
the kinetics of the P* stimulated emission and of the P+BA-
and P+BA- formation. This mode and its
overtones are apparently populated via the 130 cm-1
vibrational mode. The deuteration of the sample shifts the fundamental
frequency (32 cm-1) and all overtones by the same factor of
approximately 1.3. This mode and its overtones are suppressed by a factor of
approximately 4.7 in the dry film of RCs. The results obtained indicate that
the 32 cm-1 mode might be related to a rotation of
hydrogen-containing groups (possibly the water molecule) participating in the
modulation of the primary electron transfer from P* to BA-
in at least 35% of RCs. The Brookhaven Protein Data Bank (1PRC) displays the
water molecule located at the position HOH302 between His M200 (axial ligand
for PB) and the oxygen of ring V of BA which might be a
part (approximately 35%) of the molecular pathway for electron transfer from P*
to BA.
Yakovlev A.G., Shkuropatov A.Y., Shuvalov V.A.
Biochemistry 41 (2002) 2667-2674
Formation and
coherent propagation of nuclear wavepackets on potential energy surfaces of the
excited state of the primary electron donor P* and of the charge transfer
states P+BA- and P+HA-
were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26
reaction centers (RCs) induced by 25 fs excitation (where BA and HA
are the primary and secondary electron acceptors, respectively). The processes
were monitored by measuring coherent oscillations in kinetics of the time
evolution of the stimulated emission band of P* at 935 nm, of the absorption
band of BA- at 1020 nm, and of the bleaching band of HA
at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm-1
surface of P* is directly induced by light absorption in P. When the wavepacket
approaches the intersection between P* and P+BA-
surfaces at 120 and 380 fs delays, the formation of intermediate mixed- state
emitting light at 935 nm (P*) and absorbing light at 1020 nm (P+BA-)
takes place. At the latter time, the wavepacket is transferred to the 32 cm-1
mode which can belong to the P* hypersurface effectively transferring the
wavepacket to the P+BA- surface or can
represent adiabatic surface which is formed by the states P* and P+BA-.
The wavepacket motion on the P+BA- surface or
on the P+BA- part of the mixing surface is
accompanied by irreversible electron transfer to HA. This process is
monitored by the kinetics of 1020 nm band development and 760 nm band bleaching
(delayed with respect to 1020 nm band development) which both have the enhanced
32 cm-1 mode in Fourier transform (FT) spectra. The mechanism of wavepacket
transfer from the 130-140 cm-1 to the 32 cm-1 mode is
discussed.
Yakovlev A.G., Taisova A.S., Fetisova Z.G.
FEBS Letters 512 (2002) 129-132
It was shown
that an increase in the bacteriochlorophyll (BChl) c antenna size observed upon
lowering growth light intensities led to enhancement of the hyperchromism of
the BChl c Qy absorption band of the green photosynthetic bacterium
Chloroflexus aurantiacus. With feratosecond difference absorption spectroscopy,
it was shown that the amplitude of bleaching of the oligomeric BChl c Qy
band (as compared to that for monomeric BChl a) increased with increasing BChl
c content in chlorosomes. This BChl c bleaching amplitude was about doubled as
the chlorosomal antenna size was about trebled. Both sets of findings clearly
show that a unit BChl c aggregate in the chlorosomal antenna is variable in
size and governed by the grow light intensity, thus ensuring the high
efficiency of energy transfer within the BChl c antenna regardless of its size.
Zakharov S.D., Rokitskaya T.I., Shapovalov V.L., Antonenko Y.N., Cramer
W.A.
Proc Natl Acad Sci USA 99 (2002) 8654-8659
Membrane
surface electrostatic interactions impose structural constraints on imported
proteins. An unprecedented sensitive dependence on these constraints was seen
in the voltage-gated Import and channel formation by the C-terminal
pore-forming domain of the bacteriocin, colicin El. At physiological ionic
strengths, significant channel current was observed only in a narrow interval
of anionic lipid content ([L-1]), with the maximum current (I-max)
at 25-30 mol% (dioleoyl)- phosphatidylglycerol ([L-1]max)
corresponding to a surface potential of the lipid bilayer in the absence of
protein, Y(0)max
= -60 ± 5 mV. Higher
ionic strength shifted [L-1]max to larger values,
but Y(0)max
remained approximately constant. It is proposed that the channel current (i)
increases and (ii) decreases at Y(0) values <55 mV and >65 mV, because of (i) electrostatic
Interactions needed for effective insertion of the channel polypeptide and (ii)
constraints due to electrostatic forces on the flexibility needed for
cooperative insertion into the membrane. The loss of flexibility for Y(0) much greater than 65 mV was
demonstrated by the absence of thermally induced intraprotein distance changes
of the bound polypeptide. The anionic lipid content, 25-30 mol%, corresponding
to the channel current maxima, is similar to that of the target Escherichia
coli cytoplasmic membrane and membranes of mesophilic microorganisms. This
suggests that one reason the membrane surface potential is tuned in vivo
is to facilitate protein import.
III. MATHEMATICAL MODELS IN
BIOLOGY
Adaptive algorithm of
automated annotation
Leontovich A.M., Brodsky L.I., Drachev V.A., Nikolaev V.K.
Bioinformatics 18 (2002) 838-846
Motivation: It
is common knowledge that the avalanche of data arriving from the sequencing
projects cannot be annotated either experimentally or manually by experts. The
need for a reliable and convenient tool for automated sequence annotation is
broadly recognized. Results: Here, we describe the Adaptive Algorithm of
Automated Annotation (A4) based on a statistical approach to this
problem. The mathematical model relates a set of homologous sequences and
descriptions of their functional properties, and calculates the probabilities
of transferring a sequence description onto its homologue. The proposed model
is adaptive, its parameters (distribution characteristics, transference
probabilities, thresholds, etc.) are dynamic, i.e. are generated individually
for the sequences and various functional properties (words of the description),
The proposed technique significantly outperforms the widely used test for
frequency threshold, which is a special case of our model realized for the
simplest set of parameters. The prediction technique has been realized as a
computer program and tested on a random sequence sampling from SWISS-PROT.
IV. MOLECULAR VIROLOGY
Genome
Instability in Picornaviruses
Agol V.I.
Molecular Biology (Moscow) 36 (2002) 216-222
Picornaviruses
are small animal RNA viruses and include etiological agents of poliomyelitis,
foot and mouth disease, hepatitis A, etc. Replication of their genome results
in many mutations, which are close in number to a viability threshold. Hence
every virus population contains a great variety of genomes and represents a
quasispecies. Covalent rearrangements (deletions, insertions, recombination)
also contribute to genome variation and arise by replicative and nonreplicative
mechanisms, which are still poorly understood. Only a minor fraction of all new
changes is fixed during evolution. The fixation is based on two principally
different ways of selection: with (positive and negative selection) and without
(random selection of nonrepresentative variants) regard to the phenotype. In
natural evolution of picornaviruses, the latter way is prevalent, and most
fixed mutations are phenotypically neutral. To understand the mechanisms of
evolution, it is necessary to evaluate the biological significance of
particular genetic changes. Several new approaches to this problem have
recently been proposed.
Agol V.I.
Russian Journal of Developmental Biology 33 (2002)
279-283
The dependence
of viral reproduction and success of viral infection on cell differentiation is
briefly reviewed at the example of picornaviruses—small RNA viruses of animals.
In particular, the role of the cell factors in viral tropism, control of viral
RNA translation, and the pattern of infected cell death are discussed.
Cherkasova E.A., Korotkova E.A., Yakovenko M.L., Ivanova O.E., Eremeeva
T.P., Chumakov K.M., Agol V.I.
Journal of Virology 76 (2002) 6791-6799
Successful
implementation of the global poliomyelitis eradication program raises the
problem of vaccination against poliomyelitis in the posteradication era. One of
the options under consideration envisions completely stopping worldwide the use
of the Sabin vaccine. This strategy is based on the assumption that the natural
circulation of attenuated strains and their derivatives is strictly limited.
Here, we report the characterization of a highly evolved derivative of the
Sabin vaccine strain isolated in a case of paralytic poliomyelitis from a
7-month-old immunocompetent baby in an apparently adequately immunized
population. Analysis of the genome of this isolate showed that it is a double
(type 1-type 2-type 1) vaccine-derived recombinant. The number of mutations
accumulated in both the type 1-derived and type 2-derived portions of the
recombinant genome suggests that both had diverged from their vaccine
predecessors similar to 2 years before the onset of the illness. This fact,
along with other recent observations, points to the possibility of long-term
circulation of Sabin vaccine strain derivatives associated with an increase in
their neurovirulence. Comparison of genomic sequences of this and other evolved
vaccine-derived isolates reveals some general features of natural poliovirus
evolution. They include a very high preponderance and nonrandom distribution of
synonymous substitutions, conservation of secondary structures of important
cis-acting elements of the genome, and an apparently adaptive character of most
of the amino acid mutations, with only a few of them occurring in the antigenic
determinants. Another interesting feature is a frequent occurrence of
tripartite intertypic recombinants with either type 1 or type 3 homotypic
genomic ends.
Dorokhov Y.L., Skulachev M.V., Ivanov P.A., Zvereva S.D., Tjulkina L.G.,
Merits A., Gleba Y.Y., Hohn T., Atabekov J.G.
Proc. Natl. Acad. Sci. USA 99 (2002) 5301-5306
The internal
ribosome entry sites (IRES), IRESCP,148CR IRESMP,75,(CR) and IRE MP, precede
the coat protein (CP) and movement protein (MP) genes of crucifer-infecting
tobamovirus (crTMV), respectively. In the present work, we analyzed the
activity of these elements in transgenic plants and other organisms. Comparison
of the relative activities of the crTMV IRES elements and the IRES from an
animal virus- encephalomyocarditis virus-in plant, yeast, and HeLa cells
identified the 148-nt IRESCP,148CR as the strongest element that also displayed
IRES activity across all kingdoms. Deletion analysis suggested that the
polypurine (A)-rich sequences (PARSs) contained in IRESCP,148CR are responsible
for these features. On the basis oil' those findings, we designed artificial
PARS-containing elements and showed that they, too, promote internal
translation from dicistronic transcripts in vitro, in tobacco
protoplasts and in HeLa cells. The maximum IRES activity was obtained from
multiple copies of either A4GA2G2 or GA2-5
SCR as contained in IRESCP,148CR. Remarkably, even homopolymeric poly(A) was
moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a
database search for existing PARS sequences in T-untranslated regions (5'UTR)
of genes in tobacco genome allowed the easy identification of a number of IRES
candidates, in particular in the 5'UTR of the gene encoding Nicotiana tabacum
heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5'UTR of
NtHSF1 turned out to be an IRES element active in vitro, in plant
protoplasts and HeLa cells. We predict that PARS elements, when found in other
mRNAs, will show a similar activity.
Kalinina N.O., Rakitina D.V., Solovyev A.G., Schiemann
J., Morozov S.Y.
Virology 296 (2002) 321-329
Cell-to-cell
and long-distance transport of some plant viruses requires coordinated action
of three movement proteins encoded by triple gene block (TGB). The largest of
TGB proteins, TGBp1, is a member of the superfamily I of DNA/RNA helicases and
possesses a set of conserved helicase sequence motifs necessary for virus
movement. A recombinant His-tagged form of TGBp1 of two hordeiviruses and
potato virus X, a potexvirus, produced in Escherichia coli had unwinding
activity on a partially duplexed RNA, but not DNA substrate. The helicase
activity of these proteins was dependent on Mg2+ and ATP. The
isolated C-terminal half of the PSLV TGBp1 retaining all helicase motifs was
also able to unwind RNA duplex.
Kurganov B.I., Rafikova E.R., Dobrov E.N.
Biochemistry (Moscow) 67 (2002) 525-533
The kinetics of thermal aggregation of coat protein (CP) of tobacco
mosaic virus (TMV) have been studied at 42 and 520C in a wide range
of protein concentrations, [Plo. The kinetics of aggregation were followed by
monitoring the increase in the apparent absorbance (A) at 320 nm. At 520C
the kinetic curves may be approximated by the exponential law in the range of
TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant
being linearly proportional to Plo (50 mM phosphate buffer, pH 8.0). The
analogous picture was observed at 420C in the range of TMV CP
concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At
higher TMV CP concentrations the time of half-conversion approaches a limiting
value with increasing [P]0 and at sufficiently high protein
concentrations the kinetic curves fall on a common curve in the coordinates
(A/Alim; t) (t is time and Alim is the limiting value of
A at t --> infinity). According to a mechanism of aggregation of TMV CP
proposed by the authors at rather low protein concentrations the rate of aggregation
is limited by the stage of growth of aggregate, which proceeds as a reaction of
the pseudo-first order, whereas at rather high protein concentrations the rate-
limiting stage is the stage of protein molecule unfolding.
Development of
male reproductive organs in an insertion mutant TPD1 of tobacco characterized
by extended flowering
Milyaeva E.L., Gurko N.A., Bavrina T.V., Skurat E.V.,
Dorokhov Y.L., Romanov G.A.
Russian Journal of Plant Physiology 49 (2002) 470-477
A tobacco clone
TPD1 (Tobacco Pollen Development), characterized by an extended flowering
period, was obtained using a serial agrobacterial transformation of tobacco (Nicotiana
tabacum L., cv. Samsun) by constructing a DNA carrying the kanamycin
resistance gene inserted into the binary pBin 19 vector. However, the
characteristics of the vegetative growth of these plants were similar to those
of other tobacco clones and the wild type. In the insertion mutant, pollen was
1.5 times smaller than in the wild type and germinated on the stigmata of
neither its own clone nor the wild-type plants. Cytochemical investigation of
pollen of the TPD1-mutant did not reveal any activity of respiratory enzymes,
succinate dehydrogenase and peroxidase, indicating that the pollen was
nonviable. Unlike the wild type, the TPD1 mutant exhibited disturbed trophic
interactions within the anther tissues, particularly suppressed starch
hydrolysis in the anther wall tissues at the stage of rapidly growing
microspores. We conclude that the insertion of T-DNA into the TPD1 gene
produced structural and metabolic changes during the development of anther
tissues in the mutant clone, resulting in pollen sterility.
Zamyatnin A.A., Solovyev A.G., Sablina A.A.,
Agranovsky A.A., Katul L., Vetten H.J., Schiemann J., Hinkkanen A.E., Lehto K.,
Morozov S.Y.
Journal of General Virology 83 (2002) 651-662
The movement
function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene
block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins.
TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane
and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the
peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3
tagged with a red fluorescent protein (DsRed) was used. Coexpression of
DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP
was contained in typical ER structures and peripheral bodies formed by TGBp3
protein, suggesting an ER origin for these bodies. In transient coexpression
with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral
bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6
kDa movement protein of beet yellows closterovirus and the putative movement
protein encoded by the genome component 4 of faba bean necrotic yellows
nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the
ER membranes by artificial hydrophobic tail sequences suggested that targeting
to the ER membranes per se was not sufficient for TGBp3-directed protein
trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also
occurred in mammalian cells, indicating the universal nature of the protein
trafficking signals and the cotargeting mechanism.
V. STRUCTURE, EXPRESSION AND
EVOLUTION OF GENOM
Agapkina Yu.Yu., Agapkin D.V., Zagorodnikov A.V.,
Alekseev Ya.I., Korshunova G.A., Gottikh M.B.
Russian Journal of Bioorganic Chemistry 28 (2002)
293-299
1-( 4-( 3-(
Trifluoromethyl )-3H-diazirin-3-yl )benzamido )-3-O-( 4,4'-dimethoxytrityl
)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit
in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates
with various numbers of 2,3-propanediol moieties substituted with
aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal
stability of their duplexes with (dA)15 were studied. One ATFMD-propanediol
residue was shown to reduce the thermal stability of the duplex by 8–9°C. The
irradiation of the ATFMD-containing duplexes by UV light with the wavelength of
350 nm was found to cause the cross-linking reaction of the ATFMD-containing
strand with the complementary strand and the formation of the cross-linked
duplexes. The photomodification efficiency was independent of the
oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking.
The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1
integrase, in the presence of this enzyme resulted in the covalent DNA–protein
complex. The oligonucleotides with the
1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in
their chains can be used for the photoaffinity modification of both nucleic acids
and proteins that recognize them.
Aleshin V.V., Petrov N.B.
Zhurnal Obshchei Biologii 63 (2002) 195-208
Molecular data
permit to construct phylogenetic trees independently of morphological characters.
It allows to consider their evolution without the frames of a priori hypothesis
of regularities of morphological evolution and independently of
palaeontological. data. Cladistic analysis of elements of secondary structure
of variable areas V7 and V2 in 18S rRNA with different Protozoa as
"external" groups shows that Bilateria + Cnidaria are
monophyletic, Ctenophora and Porifera are early derivatives of Metazoa,
Trichoplax (Placozoa) is a form related to Cnidaria, while Rhombozoa,
Orthonectida and Myxozoa were branched within Bilateria.
Morphological reduction with losses of any organs and tissues took place many
times in early evolution of Metazoa and Bilateria not only in
parasitic species. It occurred both at early and late stages of embryonic development
and differentiation. Two alternative scenario of morphological degeneration in Trichoplax
and the way of their testing are suggested. The similarity of Ctenophora
and Calcarea is discussed. Meridional or oblique position of the third
cleavage furrow of ovule can be considered as an evidence of their origin from
common ancestor.
Genomics and
genosystematics
Antonov A.S.
Russian Journal of Genetics 38 (2002) 622-627
A survey of
publications dealing with comparative analysis of genomes shows that modern
genomics has naturally evolved from gene systematics- an area whose formation
and development was substantially influenced by the scientific school of A.N.
Belozersky.
Ashapkin V.V., Kutueva L.I., Vanyushin B.F.
FEBS Letters 532 (2002) 367-372
The methylation
patterns of cytosine and adenine residues in the Arabidopsis thaliana
gene for domains rearranged methyltransferase (DRM2) were studied in wild-type
and several transgene plant lines containing antisense fragments of the
cytosine DNA-methyltransferase gene METI under the control of copper-inducible
promoters. It was shown that the promoter region of the DRM2 gene is mostly
unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end
proximal part of the gene coding region is highly methylated. The DRM2 gene was
found to be also methylated at adenine residues in some GATC sequences.
Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the
DRM2 gene are variable between wild-type and different transgenic plants. The
induction of antisense METI constructs with copper ions in transgene plants in
most cases leads to further alterations in the DRM2 gene methylation patterns.
EcoRII restriction
endonuclease in catalytic conditions
Babkina O.V., Chutko C.A., Shashkov A.A., Dzhidzhoev
M.S., Eritja R., Gromova E.S.
Photochemical and Photobiological Sciences (2002)
636-640.
We used a XeCl
excimer laser with 50 ns pulses, a frequency of 0.3 Hz and a wavelength of 308
nm in appropriate conditions for the photocrosslinking of EcoRII restriction
endonuclease to a 14-mer DNA duplex, containing a 5-iodo-2'-deoxyuridine
residue (IdU). IdU replaced the thymidine residue within the EcoRII recognition
sequence 5'-CCT/AGG. The binding of EcoRII endonuclease to the IdU-containing
DNA duplex was analyzed by gel retardation assay in the presence of Ca2+ or
Mg2+ ions. Photocrosslinking of EcoRII to the IdU-containing DNA
duplex occurred in a pre-reactive complex formed in the presence of Ca2+ ions.
Photocrosslinking yields as a function of time and UV-laser light intensity
were studied.
Disruption of
HIV-1 Integrase-DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides
Brodin P., Pinskaya M., Buckle M., Parsch U., Romanova E., Engels J.,
Gottikh M., Mouscadet J.F.
Biochemistry 41 (2002) 1529-1538
We recently
found that oligonucleotides containing the 6-oxocytosine heterocyclic base are
efficient inhibitors of the HIV-1 integrase in vitro [Brodin, P., et al.
(2001) Nucleosides Nucleotides Nucleic Acids 20, 481-486]. In this report, we
demonstrate that the inhibition arises from a noncompetitive mechanism in which
the modified oligonucleotide attacks the integrase-DNA complex, leading to its
active disruption. This conclusion is based on the following results. First, despite
the fact that the respective affinities of a 6-oxocytosine-containing
oligonucleotide and of its nonmodified counterpart for integrase were
identical, only the modified compound inhibited the enzyme activities. Second,
DNA binding and UV cross-linking assays indicated that the
6-oxocytosine-containing oligonucleotide prevented the formation of a stable
integrase-DNA complex. Third, the kinetics of the dissociation of the
integrase-DNA complex were dramatically accelerated in the presence of the
modified ODN, whereas the nonmodified counterpart did not influence the
dissociation. This mechanism was supported by the ability of the
6-oxocytosine-containing oligonucleotide to inhibit the strand transfer
activity of HIV-1 preintegration complexes in vitro. Disruption of
integrase-DNA complexes by 6-oxocytosine-containing oligonucleotides
constitutes an original mechanism of integration inhibition, therefore
suggesting a strategy for searching for inhibitors of the HIV-1 preintegration
complexes.
Bunik V.I., Sievers C.
European Journal of Biochemistry 269 (2002) 5004-5015
Self-regulation
of the 2-oxo acid dehydrogenase complexes during catalysis was studied. Radical
species as side products of catalysis were detected by spin trapping, lucigenin
fluorescence and ferricytochrome c reduction. Studies of the complexes
after converting the bound lipoate or FAD cofactors to nonfunctional
derivatives indicated that radicals are generated via FAD. In the
presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide
anion radical was detected. In the absence of oxygen, a protein-bound radical
concluded to be the thiyl radical of the complex-bound dihydrolipoate was
trapped by a-phenyl-N-tert-butylnitrone.
Another, carbon-centered, radical was trapped in anaerobic reaction of the
complex with 2-oxoglutarate and CoA by 5,5'-dimethyl-1-pyrroline-N-oxide
(DMPO). Generation of radical species was accompanied by the enzyme
inactivation. A superoxide scavenger, superoxide dismutase, did not protect the
enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the
inactivation. It was concluded that the thiyl radical of the complex-bound
dihydrolipoate induces the inactivation by 1e- oxidation of the 2-oxo acid
dehydrogenase catalytic intermediate. A product of this oxidation, the
DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the
first component of the complex. The inactivation prevents transformation of the
2-oxo acids in the absence of terminal substrate, NAD+. The
self-regulation is modulated by thioredoxin which alleviates the adverse effect
of the dihydrolipoate intermediate, thus stimulating production of reactive
oxygen species by the complexes. The data point to a dual pro-oxidant action of
the complex-bound dihydrolipoate, propagated through the first and third
component enzymes and controlled by thioredoxin and the (NAD+ +
NADH) pool.
Fedoreyeva L.I., Vanyushin B.F.
FEBS Letters 514 (2002) 305-308
The N-6-adenine
DNA-methyltransferase was isolated from the vacuolar vesicle fraction of wheat
coleoptiles. In the presence of S-adenosyl-L-methionine the enzyme de novo methylates
the first adenine residue in the TGATCA sequence in the single- or
double-stranded DNA substrates but it prefers single-stranded structures. Wheat
adenine DNA-methyltransferase (wadmtase) is a Mg2+- or Ca2+-dependent
enzyme with a maximum activity at pH 7.5-8.0. Wadmtase seems to be responsible
for mitochondrial DNA modification that might be involved in the regulation of
replication of mitochondria in plants.
Filippova S.E., Ivanovskaya M.G., Romanova E.A., Tunitskaya V.L.,
Kochetkov S.N.
Molecular Biology 36 (2002) 543-550
The contacts
between phosphate groups of promoter DNA and Lys or His of T7 RNA polymerase
(Pot) in the Pot-promoter complex were studied with single- and double-stranded
oligonucleotides corresponding to the T7 promoter consensus and containing
activated phosphate groups at position +1, +2, or -14 relative to the
transcription start. To obtain reactive groups, terminal phosphates were
modified with N-hydroxybenzotriazole (HOBT), and internucleotide phosphates
were replaced with a trisubstituted pyrophosphate (TSP). The resulting
derivatives produced covalent complexes with T7 Pot. Covalent bonding involved
His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in
the case of TSP at position -14.
Gulevich A.Y., Yusupova R.A., Drygin Y.F.
Biochemistry (Moscow) 67 (2002) 615-621
VPg unlinkase
is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester
bond between residues of the unique tyrosine of VPg (viral protein
genome-linked) and the 5'-terminal uridylic acid of picornavirus RNA. Cellular
targets of the VPg unlinking enzyme are yet unknown. To determine an essential
nucleic pan of the covalent linkage unit that is necessary for the VPg
unlinkase reaction, the following derivatives of the encephalomyocarditis virus
(EMCV) VPg-RNA complex were used: [I-125]Kp-pUpUpGp, [I-125]Kp-pUp, and
[I-125]Kp-pU (Kp is residual peptides bound to RNA after proteinase K treatment
of VPg-RNA). [I-125]K-peptides were unlinked from [ 1251]Kp-pUpUpGp and
[I-125)Kp-RNA with similar velocity, but [I-125]Kp-pUp was split much slower.
Under the same conditions [125 I]Kp-pU was not dissociated at all. Thus, pUp is
a minimal pan of picornavirus RNA that is necessary for VPg unlinkase. We
speculate that cellular substrates of the enzyme are phosphodiesters of
oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case
[I-125]Vpg-pU, [I- 125]VPg-pUp, and [I-125]VPg-pUpUpGp were hydrolyzed by VPg
unlinkase, in contrast with [I-125]VPg-RNA and [I-125]VPg- pUpUpGpApApApGp. We
conclude that the whole VPg, when bound to trinucleotide (but not to
heptanucleotide), protects the inter- polymeric phosphodiester bond against
hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might
repair covalent complexes of RNA and topoisomerases and trigger degradation
process of the picornavirus RNA.
Hehlgans T., Stoelcker B., Stopfer P., Muller P., Cernaianu G., Guba M.,
Steinbauer M., Nedospasov S.A., Pfeffer K., Mannel D.N.
Cancer Research 62 (2002) 4034-4040
Growth of solid
fibrosarcoma tumors in mice was inhibited by the release of a soluble
lymphotoxin-b receptor
inhibitor (LTbR-immunoglobulin
fusion protein) from the tumor cells. Tumor growth arrest in mice deficient in
the ligand LTa(1)b(2) demonstrated the requirement for
activation of the LTbR on the tumor
cells by host cell-derived LTa(1)b(2). Activation
of the LTbR resulted in
enhanced release of macrophage inflammatory protein-2. Blocked angiogenesis was
revealed in LTbR
inhibitor-producing tumor nodules by immunohisto-chemistry and in vivo
microscopy. The growth arrest of LTbR inhibitor-producing fibrosarcomas was overcome by forced MIP-2
expression in the tumor cells. Thus, LTbR activation on tumor cells by
activated host lymphocytes can initiate a novel proangiogenic pathway leading
to organized tumor tissue development.
Ivanov S.A., Alekseev Y.I., Gottikh M.B.
Molecular Biology 35 (2002) 131-139
Interactions of
oligonucleotides comprising ( 1-b-D-2'-deoxy- threo-pentafuranosyl )thymine and (1-b-D-2'-deoxy-threo- pentafuranosyl)cytosine
residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded
DNA fragments were investigated. Using nondenaturing gel electrophoresis,
footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes
with the purine DNA template, which are stable at neutral pH and comparable in
heat stability with the corresponding natural polypurine-polypyrimidine DNA
duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are
protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of
the triple helix depends on the nucleotide composition and sequence of the DNA
template, and is intermediate between A and B.
Kachalova A.V., Stetsenko D.A., Romanova E.A.,
Tashlitsky V.N., Gait M.J., Oretskaya T.S.
Helvetica Chimica Acta 85 (2002) 2409-2416
An efficient
method for synthesis of oligonucleotide 5'- conjugates through amide-bond formation
on solid phase is described. Protected oligonucleotides containing a 5'-
carboxylic acid function were obtained by use of a novel non- nucleosidic
phosphoramidite building block, where the carboxylic acid moiety was protected
by a 2-chlorotrityl group. The protecting group is stable to the
phosphoramidite coupling conditions used in solid-phase oligonucleotide
assembly, but is easily deprotected by mild acidic treatment. The protecting
group may be removed also by ammonolysis. 5'-Carboxylate- modified
oligonucleotides were efficiently conjugated on solid support under normal
peptide-coupling conditions to various amines or to the N-termini of small
peptides to yield products of high purity. The method is well-suited in
principle for the synthesis of peptide-oligonucleotide conjugates containing an
amide linkage between the 5'-end of an oligonucleotide and the N-terminus of a
peptide.
Kalmykova A.I., Shevelyov Y.Y., Polesskaya O.O.,
Dobritsa A.A., Evstafieva A.G., Boldyreff B., Issinger O.G., Gvozdev V.A.
European Journal of Biochemistry 269 (2002) 1418-1427
An earlier
described CK2betates gene of Drosophila melanogaster is shown to encode
a male germline specific isoform of regulatory beta subunit of casein kinase 2.
Western-analysis using anti-CK2betates Ig revealed CK2betates protein in Drosophila
testes extract. Expression of a CK2betates-b- galactosidase fusion protein driven
by the CK2betates promoter was found in transgenic flies at postmitotic stages
of spermatogenesis. Examination of biochemical characteristics of a recombinant
CK2betates protein expressed in Escherichia coli revealed properties
similar to those of CK2b: (a) CK2betates protein stimulates CK2a catalytic activity toward synthetic
peptide: (b) it inhibits phosphorylation of calmodulin and mediates stimulation
of CK2a by polylysine: (c) it is able to form (CK2betates)2 dimers,
as well as (CK2a)2(CK2betates)2
tetramers. Using the yeast two- hybrid system and coimmunoprecipitation
analysis of protein extract from Drosophila testes. we demonstrated an
association between CK2betates and CK2a. Northern-analysis has shown that another regulatory (beta') subunit found
recently in D. melanogaster genome is also testis-specific. Thus, we
describe the first example of two tissue-specific regulatory subunits of CK2
which might serve to provide CK2 substrate recognition during spermatogenesis.
Kopylov A.M.
Biochemistry (Moscow) 67 (2002) 372-382
This review
considers a brief history, comments, and consequences of recent remarkable
achievements: X-ray analysis on the level of atomic resolution of structures of
bacterial ribosomes, their subunits, and functional complexes.
Koudan E.V., Subach O.M., Korshunova G.A., Romanova E.A., Eritja R.,
Gromova E.S.
Journal of Biomol. Struct. Dyn. 20 (2002) 421-428
EcoRII DNA
methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and
catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the
C5 position of the inner cytosine residue (C*). We obtained several DNA
duplexes containing photoactive 5-iodo-2'-deoxyuridine (i5dU) or
5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU)
to characterize regions of M.EcoRII involved in DNA binding and to investigate
the DNA double helix conformational changes that take place during methylation.
The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA
photocrosslinking yields strongly depend on the type of modification and its
location within the EcoRII recognition site. The data obtained agree with the
flipping of the target cytosine out of the DNA double helix for catalysis. To
probe regions of M.EcoRII involved in DNA binding, covalent conjugates
M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the
oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU
at the central position of the recognition site, or instead of the target
cytosine were crosslinked to the Gly268-Met391 region of
the EcoRII methylase. Amino acid residues from this region may take part both
in substrate recognition and stabilization of the extrahelical target cytosine
residue.
Kuprash D.V., Boitchenko V.E., Yarovinsky F.O., Rice N.R., Nordheim A.,
Ruhlmann A., Nedospasov S.A.
Blood 100 (2002) 1721-1727
The 2
lymphotoxin subunits LTa (also called tumor necrosis factor b [TNF-b]) and LTb belong to the family of TNF-related
cytokines. They form either a soluble homotrimeric ligand (LTa(3)) that binds to and signals
through CD120a/b (TNFRp55 and TNFRp75), or a membrane-associated heterotrimeric
ligand (LTa(1)b(2)) that binds to and signals
through the LTb receptor (LTbR). In mice, LTbR signaling is critical for the
maintenance of peripheral lymphoid tissues and optimal immune responses, and
its down-regulation results in immunodeficiency. To determine the possible
relationship between LT-mediated immunodeficiency and the immunosuppressive
effects of cyclosporin A (CsA), we tested the effects of CsA on the expression
of LTa and LTb in human peripheral blood mononuclear
cells (PBMCs). When PBMCs were stimulated with phorbol myristate
acetate/ionomycin or with anti- CD3/anti- CD28, the accumulation of LTa both at mRNA and protein levels was
markedly inhibited by CsA. This inhibition is likely due to CsA's effect on the
nuclear factor of activated T cell (NFAT) proteins binding to a novel
NFAT-binding element at position -490 relative to LTa transcription start. LTb showed a distinct expression
pattern and was insensitive to CsA. Thus, in addition to its effects on the
expression of other TNF family members, such as TNFa, CD40-L, and CD95-L, CsA can block
expression of surface LT complex by selectively inhibiting the expression of
the LTa subunit. We
propose that LT dysfunction and its downstream effects may contribute to
immunosuppressive effects of CsA.
Kubareva E.A., Walter J., Karyagina A.S., Vorob'eva O.V., Lau P.C.,
Trautner T.
Biotechniques 33 (2002) 526-531
Using a new
method based on a combination of bisulfite reaction, the repair enzyme
uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the
methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria
lactamica was established to be the inner cytosine in the double-stranded
pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside).
5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of
oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.
Kuvardina O.N., Leander B.S., Aleshin V.V., Myl'nikov A.P., Keeling
P.J., Simdyanov T.G.
Journal of Eukaryot. Microbiol. 49 (2002) 498-504
In an attempt
to reconstruct early alveolate evolution, we have examined the phylogenetic
position of colpodellids by analyzing small subunit rDNA sequences from Colpodella
pontica Myl'nikov 2000 and Colpodella sp. (American Type Culture
Collection 50594). All phylogenetic analyses grouped the colpodellid sequences
together with strong support and placed them strongly within the Alveolata.
Most analyses showed colpodellids as the sister group to an apicomplexan clade,
albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera,
clustered together and consistently branched as the sister group to
dinoflagellates as shown previously. These data demonstrate that colpodellids
and perkinsids are plesiomorphically similar in morphology and help provide a
phylogenetic framework for inferring the combination of character states
present in the last common ancestor of dinoflagellates and apicomplexans. We
can infer that this ancestor was probably a myzocytotic predator with two
heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar
ring, and a coiled open-sided conoid. This ancestor also very likely contained
a plastid, but it is presently not certain whether it was photosynthetic, and it
is not clear whether extant perkinsids or colpodellids have retained the
organelle.
Kuznetsov G.V., Kulikov E.E., Petrov N.B., Ivanova
N.V., Lomov A.A., Kholodova M.V., Poltaraus A.B.
Molecular Phylogenetics and Evolution 23 (2002) 91-94
The
phylogenetic position and taxonomic status of the recently described Southeast
Asian endemic bovid Pseudonovibos spiralis were deduced from nearly
complete 12S mitochondrial rDNA sequences of this species and Bubalus
bubalis alongside 26 sequences of Bovidae from GenBank using Cervus
elaphus (Cervidae) as outgroup. Maximum-likelihood analyses
performed by PUZZLE and fastDNAml nested P. spiralis at the base of the
subtribe buffalo Bovini, suggesting the close relationship of this
enigmatic species with buffalo and enabling its distinction into the separate
subtribe Pseudonovibovina.
Kuznetsova M.V., Kholodova M.V., Luschekina A.A.
Russian Journal of Genetics 38 (2002) 942-950
The phylogeny
of the family Bovidae has been inferred from our data on the 12S and 16S
rRNA mtDNA gene sequences and from the results of other authors. A considerable
(2460 bp) length of the analyzed fragments of these conserved genes and the use
of different methods of cladogram construction allowed us to verify the
systematic position of the genera Saiga, Pantholops, Procapra,
and Oreamnos. Saigas were shown to be phylogenetically far closer to
gazelles than black-tailed gazelles and pygmy antelopes. In general, the
genetic analysis data are in agreement with the results of morphological
studies.
Ledneva R.K., Alexeevskii A.V., Vasil’ev S.A., Spirin
S.A., Karyagina A.S.
Molecular Biology (Moscow) 35 (2001) 647-659
This review is
devoted to the structural aspects of interaction of homeodomains with DNA.
Presented are the list of all homeodomains with known spatial structure and the
alignment of their amino acid sequences. The structure of homeodomains and
contacts of their amino acid residues with DNA bases and sugar-phosphate
backbone are described. The role of water molecules in DNA binding is
discussed. Structures of multicomponent protein complexes on DNA including
homeodomains are characterized.
Lukashina E.V., Badun G.A., Ksenofontov A.L., Baratova L.A., Dobrov
E.N., Fedoseev V.M.
Radiochemistry 44 (2002) 81-85
The possibility
of measuring a low activity of tritium-labeled amino acids in the eluate from
Amino Acid Analyzer 835 (Hitachi, Japan) using a Radiomatic 150TR Flow
Scintillation Analyzer (Packard Instrument Co., USA) was studied. Due to
stepped variations of pH, ionic strength, and salt concentration in eluting
solutions during amino acid separation and utilization of ninhydrin reagent in
spectrophotometric measurements of amino acids, special selection of
scintillation liquids was necessary. Six scintillation cocktails were tested:
ZhS-8 (Reakhim, Ukraine), OptiPhase “HiSafe” 3 (Wallac Oy, EG&G Co.,
Finland), Hionic-Fluor, Ultima-Flo AP, Ultima-Flo M, and Ultima Gold (Packard
Instrument Co., USA). It was found that Hionic-Fluor and Ultima-Flo AP
cocktails are the most appropriate for flow measurements of tritium activity.
Under optimal conditions the detection limit with Hionic-Fluor and Ultima-Flo
AP was 150 and 100 decays min-1 in the peak of amino acid,
respectively. Such a high sensitivity allows utilization of the above
analytical system for measurements of amino acid radioactivity to study the
structure of proteins and protein complexes by tritium planigraphy.
Luzikov V.N.
Biochemistry (Moscow) 67 (2002) 171-183
This review
summarizes materials on the mechanisms of intracellular degradation of proteins
whose topogenesis is disturbed at one stage or another. Chaperone and
proteolytic systems involved in this process in the endoplasmic reticulum,
mitochondria, and chloroplasts of eucaryotic cells as well as those in distinct
subcellular compartments of procaryotic cells are considered. The available
data suggest that living cells contain numerous systems keeping under control
both folding of newly synthesized and newly imported polypeptide chains and
their incorporation into heterooligomeric complexes. The point of view is
elaborated that organelle formation is controlled not only at the level of
individual protein molecules but also at the supermolecular level when whole
organelles incapable of carrying out their integral key functions become
targets for partial or total elimination. This type of control is realized
through an autophagic mechanism involving lysosomes/vacuoles.
Oretskaya T.S., Romanova E.A., Andreev S.Y., Antsypovich S.I., Toth C.,
Gajdos V., Hianik T.
Bioelectrochemistry 56 (2002) 47-51
An effective
method for the introduction of oleylamine-modified cytidine units into
predetermined position(s) of the oligodeoxyribonucleotide (ON) chain during
automated ON synthesis has been developed. The high yields of the condensation
products upon the introduction of the modified units allow the methods
suggested to be used for the synthesis of ONs with two hydrophobic
substituents. We also suggest a simple method for obtaining ONs with
5'-terminal hydrophobic linker with free thiol group. The functionality of
synthesized ON modified by thiol group and that with hydrophobic spacer for the
detection DNA hybridization has been approved in conductometric experiments.
Pavlov N.A., Cherny D.I., Heim G., Jovin T.M., Subramaniam V.
FEBS Letters 517 (2002) 37-40
Mammalian
prothymosin a, a small (12 kDa) and extremely acidic protein (pI 3.5), is a
member of the growing family of `natively' unfolded proteins. We demonstrate
that at low pH ( similar to 3) and high concentrations, prothymosin a is
capable of forming regular elongated fibrils with flat ribbon structure 4-5 nm
in height and 12-13 nm in width as judged from scanning force and electron
microscopy. These aggregates induced a characteristic spectral shift of thioflavin
T fluorescence and their circular dichroism spectra were indicative of
significant b-sheet content,
suggesting formation of classical amyloid. Our findings indicate that natively
unfolded proteins may have a general propensity to form amyloid fibrils under
conditions inducing partially folded conformations.
Pavlov N.A., Cherny D.I., Nazimov I.V., Slesarev A.I., Subramaniam V.
Nucleic Acids Research 30 (2002) 685-694
Three novel DNA-binding proteins with apparent molecular masses of 7, 10
and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus
kandleri. The proteins were identified using a blot overlay assay that was
modified to emulate the high ionic strength intracellular environment of
M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia
coli. As indicated by CD spectroscopy and computer-assisted structure
prediction methods, 7kMk is a substantially a-helical protein possibly containing
a short N-terminal b-strand. According to analytical gel filtration chromatography and
chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further
oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads
to the formation of loop-like structures of similar to 43.5±3.5 nm (136±11 bp for B-form DNA) circumference.
A topoisomerase relaxation assay demonstrated that looped DNA is negatively
supercoiled under physiologically relevant conditions (high salt and
temperature). A BLAST search did not yield 7kMk homologs at the amino acid
sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH)
transcriptional regulators, fold features and self-association properties of
7kMk we hypothesize that it could be related to RHH proteins.
Petrov N.B., Aleshin V.V.
Russian Journal of Genetics 38 (2002) 877-894
The current
phase of molecular phylogenetics can be named the 18S rRNA gene era, which is
now approaching the end. To date, almost all phyla of metazoans and many taxa
of protists are represented in databases of 18S rRNA gene sequences. The
elements of the phylogenetic tree of Metazoa inferred from 18S rRNA
genes are characterized by unequal validity: some of them seem to be well
grounded; others are not adequately supported, and probably will be revised
later. The validity of phylogenetic reconstruction is influenced by two main
factors: (1) erroneous grouping of long branches that occur because of
abnormally high evolution rate; (2) deficit of phylogenetically informative
characters. A method for overcoming these difficulties is suggested in addition
to known tools: using phylogenetic markers that are stable within individual
taxa and evolve by punctuated equilibrium. These markers are least influenced
by the convergence caused by a high evolution rate of the entire gene. The
nature of these markers of ancient taxa, paradoxical from the perspective of
neutral evolution, is discussed, as well as their importance for establishing
monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria
+ Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa
and Echinodermata + Hemichordata) and some major taxa of Nematoda.
Pingoud V., Kubareva E., Stengel G., Friedhoff P., Bujnicki J.M.,
Urbanke C., Sudina A., Pingoud A.
Journal of Biological Chemistry 277 (2002) 14306-14314
The type II
restriction endonuclease SsoII shows sequence similarity with 10 other
restriction endonucleases, among them the type HE restriction endonuclease
EcoRII, which requires binding to an effector site for efficient DNA cleavage,
and the type IIF restriction endonuclease NgoMIV, which is active as a
homotetramer and cleaves DNA with two recognition sites in a concerted
reaction. We show here that SsoII is an orthodox type II enzyme, which is
active as a homodimer and does not require activation by binding to an effector
site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding
site and catalytic center as shown here by a mutational analysis, indicative of
an evolutionary relationship between these three enzymes. We suggest that a
similar relationship exists between other orthodox type II, type IIE, and type
IIF restriction endonucleases. This may explain why similarities may be more
pronounced between members of different subtypes of restriction enzymes than
among the members of a given subtype.
Pisarev A.V., Skabkin M.A., Thomas A.A., Merrick W.C., Ovchinnikov L.P.,
Shatsky I.N.
Journal of Biological Chemistry 277 (2002) 15445-15451
AB p50, the
major core protein bound to mammalian mRNAs, has been reported to stimulate
translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA
ratios. This study aims to address the molecular mechanisms underlying these
phenomena using the in vitro assembly of 48 S preinitiation complexes
from fully purified translational components in the presence or absence of p50
as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3,
and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for
comparison) strongly stimulates formation of the 48 S preinitiation complexes
with b-globin mRNA.
This stimulation is observed when just a few molecules of p50 are bound per
molecule of the mRNA. When the amount of p50 in solution is increased over some
threshold p50/mRNA ratio, a remarkable repression is observed that can still be
relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50,
the inhibitory effect becomes irreversible. The threshold ratio depends upon
the extent of secondary structure of the 5'- untranslated region linked to the
P-globin coding region. Chemical probing has confirmed that the binding of p50
to mRNA involves only the sugar-phosphate backbone of the mRNA leaving
nucleotide bases free for interaction with other messenger ribonucleoprotein
(mRNP) components. These data are best compatible with the functional role of
p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.
Smirnova E.G., Lyubimov Yu.I., Malinina T.G., Lyubimova E.Yu.,
Alexandrushkina N.I., Vanyushin B.F., Kolesova G.M., Yaguzhinsky L.S.
Biochemistry (Moscow) 67 (2002) 1271-1275
In aqueous
medium etiolated wheat seedlings release superoxide anion (Î2-) Interaction of
a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in
the aqueous medium is accompanied by Î2 -formation. This suggests that under
certain conditions BHT behaves as a prooxidant. A natural antioxidant,
superoxide dismutase (SOD), and also a wound healing preparation, emulsified
denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast
to BHT, they reduce Î2 production by the etiolated wheat seedling system.
Spiridonova V.A., Kopylov A.M.
Biochemistry (Moscow) 67 (2002) 706-709
A fiber-optic
biosensor based on DNA aptamers used as receptors was developed for the
measurement of thrombin concentration. Anti-thrombin DNA aptamers were
immobilized on silica microspheres, placed inside microwells on the distal tip
on an imaging optical fiber, coupled to a modified epifluorescence microscope
through its proximal tip. Thrombin concentration is determined by a competitive
binding assay using a fluorescein- labeled competitor. The biosensor is
selective and can be reused without any sensitivity change. The thrombin limit
of detection is 1 nM, Sample Volume is 10 ?l, and assay time per sample is 15 min including the regeneration step.
Spiridonova V., Rassokhin T., Golovin A., Petrova E., Rohzdestvensky T.,
Pakhomova Y., Kopylov A.
Bioelectrochemistry 56 (2002) 95-97
In recent
years, Systematic Evolution of Ligands by EXponential enrichment (SELEX)
technique has been developed into a fast growing field. In contrast to
traditional recognition elements, like antibody, our interests focus on novel
molecular recognition elements based on nucleic acids, which are of value both
for the therapy and biosensors. A comparative study of thermodynamic for both
natural and artificial RNA/DNA-protein complexes would establish bases for a
specificity of complex formation. In particular, we have shown that aptamers
could be used for a direct measuring of thrombin enzymatic activity in a
solution.
Sukhacheva E.A., Evstafieva A.G., Fateeva T.V., Shakulov V.R., Efimova
N.A., Karapetian R.N., Rubtsov Y.P., Vartapetian A.B.
Journal of Immunological Methods 266 (2002) 185-196
To overcome
poor immunogenicity of prothymosin a, a small and highly acidic nuclear
protein involved in cell proliferation, production of anti-prothymosin a antibodies in mice immunized with
free human prothymosin a, with prothymosin a coupled to different carriers and with prothymosin a fused to green fluorescent protein
was assessed. Fusing prothymosin a to green fluorescent protein turned out to be the superior approach
resulting in production of high titer anti-prothymosin a antibodies. From these studies, two
highly specific anti-prothymosin a monoclonal antibodies recognizing epitopes within the amino terminal
(2F11) and middle (4F4) portions of the human prothymosin a molecule were obtained and
characterized. As expected, the 2F11 antibody displayed broad species
specificity, whereas the 4F4 antibody appeared to be species-specific
permitting discrimination of human versus rat protein. Furthermore, a
combination of point mutations in prothymosin a that alter the properties of the
protein precluded recognition by the 4F4 antibody. Intramolecular masking of
the 4F4 epitope in prothymosin a fused to the Tat transduction peptide of human immunodeficiency virus
type 1 was observed. The anti- prothymosin a antibodies obtained were suitable
for precipitation of human prothymosin a from HeLa cell lysates and for
immunolocalization of the endogenous prothymosin a within the cells. Fusion with green
fluorescent protein may thus be helpful in raising antibodies against
'problematic' proteins.
Distinct role of
surface lymphotoxin expressed by B cells in the organization of secondary
lymphoid tissues
Tumanov A.V., Kuprash D.V., Lagarkova M.A.,
Grivennikov S.I., Abe K., Shakhov A.N., Drutskaya L.N., Stewart C.L.,
Chervonsky A.V., Nedospasov S.A.
Immunity 17 (2002) 239-250.
In order to
definitively ascertain the functional contribution of lymphotoxin (LT) expressed
by B cells, we produced mice with the LTb gene deleted from B cells (B-LTb KO mice). In contrast to systemic
LTb deletion, in
B-LTb KO mice only
splenic microarchitecture was affected, while lymph nodes and Peyer's patches
(PP) were normal, except for PP's reduced size. Even though B-LTb KO spleens retained a small number
of follicular dendritic cells (FDC) which appeared to be dependent on LPb produced by T cells, IgG responses
to sheep red blood cells were markedly reduced. Thus, the organogenic function
of B-LTb is almost
entirely restricted to spleen, where it supports the correct lymphoid
architecture that is critical for an effective humoral immune response.
Valiejo-Roman C.M., Terentieva E.I., Samigullin T.H.,
Pimenov M.C.
Taxon 51 (2002) 91-101
The internal
transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were newly
sequenced for eight species of Umbelliferae (six species from subfamily Saniculoideae:
Actinolema macrolema, Astrantia minor, Eryngium giganteum,
E. coeruleum, Hacquetia epipactis, and Lagoecia cuminoides, two
species from subfamily Hydrocotyloideae: Dickinsia hydrocotyloides and Azorella
trifurcata), as well as Hohenackeria exscapa, a species of uncertain
position in the family. Phylogenetic analyses of new data, plus previously
reported sequences of 52 other species using neighbor-joining, maximum
parsimony, and maximum likelihood methods yielded similar results: (1) Actinolema
is sister to Astrantia corresponding to Drude's treatments; (2) in Astrantia,
molecular divergence is revealed between sects. Astrantiella (A. minor)
and Astrantia (A. major, A. maxima); (3) Eryngium appears
to be paraphyletic; (4) Hacquetia might be treated as a part of Sanicula;
and (5) Lagoecia is very distant from all other Saniculoideae and
close to some genera of Apioideae. Our results correspond to matK data
previously published: (1) Hohenackeria forms a clade with Bupleurum,
in a position near the base of the Apioideae tree; (2) Azorella
is sister to a large cluster uniting all Saniculoideae and Apioideae,
being slightly closer to them than to the Hydrocotyle-Araliaceae
clade; (3) Dickinsia is very distant from phenetically similar Hydrocotyle,
falling within a large cluster of Apioideae, but also including Lagoecia
and Naufraga.
Vanyushin B.F., Shorning B.Y., Seredina A.V., Aleksandrushkina N.I.
Russian Jorrnal of Plant Physiology 49 (2002) 501-506
The development
of etiolated wheat (Triticum aestivum L.) seedlings is necessarily
accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome
DNA fragmentation, which is characteristic of apoptosis, was detected in the
coleoptile as soon as six days after germination. After eight days of
germination, DNA fragmentation was clearly expressed in the coleoptile and was
noticeable in the apical part of the first-leaf blade. Growing of intact
seedlings or incubation of their shoots in the presence of such phytohormones
as benzyladenine, gibberellin A3, fusicoccin C, and 2,4-D at the
concentration of 10-5 M did not essentially affect DNA fragmentation
in the coleoptile. As distinct from antioxidants, none of the phytohormones
used prevented apoptosis in wheat seedlings. In contrast, ABA (10-5
M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10-2-10-3
M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA
methylation, 5-azacytidine, was very efficient in the stimulation of DNA
fragmentation in the coleoptiles of eight-day-old seedlings at its
concentration of 100 ?g/ml. Thus, some
phytohormones can regulate apoptosis, and DNA methylation is involved in this
process. Our results indicate that apoptosis activation by some phytohormones
may be mediated by their regulation of DNA methylation/demethylation, which is
responsible for the induction of genes encoding apoptogenic proteins and/or the
repression of antiapoptotic genes.
Zamyatnina V.A., Bakeeva L.E., Aleksandrushkina N.I., Vanyushin B.F.
Biochemistry (Mjscow) 67 (2002) 67 212-221
Apoptosis was
observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A
condensation of cytoplasm in apoptotic cells, formation of myelin-like
structures, specific fragmentation of cytoplasm, appearance in vacuoles of
specific vesicles containing subcellular organelles, condensation and
margination of chromatin in the nucleus, and internucleosomal fragmentation of
nuclear DNA are ultrastructural features of apoptosis in the initial wheat
leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble
in appearance the vacuolar vesicles in the coleoptile apoptotic cells described
earlier (Bakeeva, L. E., et at. (1999) FEBS Lett., 457, 122-125); they contain
preferentially plastids but not mitochondria as was observed in coleoptile. The
vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis
in various tissues is an obligatory element of plant (wheat) growth and
development even in the early stages of ontogenesis. Contrary to strong
geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27 x
10-4 M) does not prevent in the leaf cells the apoptotic
internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles
containing subcellular organelles. Therefore, the antioxidant action on
apoptosis in plants is tissue specific. Peroxides (H2O2,
cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation)
in coleoptile and induced it in an initial leaf when apoptosis in a control
seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant
ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by
anti- and prooxidants.
Apoptosis in
Etiolated Wheat Seedlings: 1. Ultrastructure of the Apoptotic Cell
Zamyatnina V.A., Bakeeva L.E., Aleksandrushkina N.I.,
Vanyushin B.F.
Russian Journal of Plant Physiology 49 (2002) 736-745
We studied the
process of apoptosis in etiolated wheat (Triticum aestivum L.) seedlings.
As a result, an integral pattern of the apoptotic plant cell ultrastructure was
established. In the apoptotic cells of the coleoptile, we observed chromatin
condensation and margination, an increased density and specific cytoplasm
fragmentation accompanied by the appearance of unusual cytoplasmic vesicles
containing subcellular organelles, mitochondria in particular, in the vacuoles.
Zatsepin T.S., Romanova E.A., Oretskaya T.S.
Uspekhi Khimii 71 (2002) 586-608
Methods for the
synthesis of 2'-O-alkylnucleosides are reviewed. The known methods are
described systematically, their advantages and drawbacks are discussed.
Zatsepin T.S., Stetsenko D.A., Arzumanov A.A.,
Romanova E.A., Gait M.J., Oretskaya T.S.
Bioconjugate Chemistry 13 (2002) 822-830
2'-Deoxyoligonucleotides
and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups
were synthesized and coupled to peptides containing an N-terminal cysteine,
aminooxy, or hydrazide group to give peptide-oligonucleotide conjugates
incorporating single or multiple peptides in good yield. The facile conjugation
method allows specific coupling in aqueous solution of unprotected
oligonucleotides containing aldehyde groups to unprotected N-terminally
modified peptides and other small molecules. A 12-mer 2'-O- methyloligoribonucleotide
complementary to the HIV-1 TAR RNA stem-loop and containing two conjugated
copies of an 8-mer model laminin peptide was hardly affected in TAR RNA binding
and showed a similar level of inhibition of HIV-1 Tat-dependent in vitro
transcription compared to the unconjugated 2'-O- methyloligoribonucleotide.
Advantages of this conjugation method include (1) the ability to attach more
than one peptide or other small molecule to oligonucleotide at defined
nucleoside residue locations; (2) a conjugation route that does not affect
significantly oligonucleotide binding to RNA structures; and (3) three
alternative, facile, and mild conjugation reaction types that do not require
use of a large excess of peptide reagent.
Zhirnov O.P., Ksenofontov A.L., Kuzmina S.G., Klenk H.D.
Biochemistry (Moscow) 67 (2002) 534-539
In this
investigation, an ability of influenza A virus M1 matrix protein to bind
intracellular caspases, the key enzymes of cell apoptosis, has been examined.
Protein-protein binding on polystyrene plates and polyvinyl pyrrolidone
membrane was employed for this purpose. Under a comparative study of
caspases-3, -6, -7, -8 influenza virus M1 protein specifically bound caspase-8 and
weakly bound caspase-7. Using a computer analysis of the N-terminal region of
M1 protein, a site similar to the anti-caspase site of baculovirus p35 protein,
which inhibits caspases and displays antiapoptotic activity, was identified.
These results are in good agreement with the supposition that influenza virus
M1 protein is involved in a caspase-8-mediated apoptosis pathway in influenza
virus infected cells.
Zubin E.M., Romanova E.A., Oretskaya T.S.
Uspekhi Khimii 71 (2002) 273-302
Published data
on the methods of chemical synthesis of peptide- oligonucleotide conjugates in
solution and on the solid phase are discussed. The known methods are systematised,
their advantages and drawbacks are noted. The studies devoted to the synthesis
of peptide-oligonucleotide conjugates in solution are arranged according to the
type of chemical band between the fragments, while in the case of solid-phase
synthesis, according to the method of conjugate preparation (either successive
growth of the oligonucleotide and peptide chains on a single polymer support or
solid-phase condensation of two presynthesised fragments).
VI.
ENZYMOLOGY AND BIOTECHNOLOGY
Belogurov G.A., Turkina M.V., Penttinen A., Huopalahti S., Baykov A.A.,
Lahti R.
Journal of Biological Chemistry 277 (2002) 22209-22214
H+-translocating
pyrophosphatase (H+-PPase) of the photosynthetic bacterium Rhodospirillum
rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant
H+-PPase was observed in inner membrane vesicles, where it catalyzed
both PPi hydrolysis coupled with H+ transport into the
vesicles and PPi synthesis. The hydrolytic activity of H+-PPase
in E. coli vesicles was eight times greater than that in R. rubrum
chromatophores but exhibited similar sensitivity to the H+-PPase
inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase
inhibitor, fluoride. Using this expression system, we showed that substitution
of Cys185, Cys222, or Cys573 with aliphatic
residues had no effect on the activity of H+-PPase but decreased its
sensitivity to the sulfhydryl modifying reagent, mersalyl. H+-PPase
lacking all three Cys residues was completely resistant to the effects of
mersalyl. Mg2+ and MgPPi protected Cys185 and
Cys573 from modification by this agent but not Cys222.
Phylogenetic analyses of 23 nonredundant H+-PPase sequences led to
classification into two subfamilies. One subfamily invariably contains Cys222
and includes all known K+-independent H+-PPases, whereas
the other incorporates a conserved Cys573 but lacks Cys222
and includes all known K+-dependent H+-PPases. These data
suggest a specific link between the incidence of Cys at positions 222 and 573
and the K+ dependence of H+-PPase.
Chilov G.G., Svedas V.K.
Canadian Journal of Chemistry – Revue Canadienne de Chimie 80 (2002)
699-707
The application
of the two-phase "aqueous solution - water- immiscible organic
solvent" system is suggested not for effective biocatalytic synthesis, but
for hydrolytic purposes. Enzymatic hydrolysis of benzylpenicillin and
N-phenylacetamidodesacetoxycephalosporanic acid to corresponding antibiotic
nuclei 6-aminopenicillanic and 7- aminodesacetoxycephalosporanic acids in a
two-phase water-butyl acetate system at pH 3-4 is proposed as an alternative to
the biocatalytic hydrolysis in an alkaline medium. An experimental study has
been performed and a model has been developed, which describes the influence of
pH, phase volume ratio, thermodynamic constants, and initial antibiotic
concentration on the effectiveness of their hydrolysis in a two-phase
"aqueous solution - water-immiscible organic solvent" system. The
thermodynamic evaluation of penicillin G and 7-
phenylacetamidodesacetoxycephalosporanic acid hydrolysis at low pH in a
two-phase aqueous solution - water-immiscible organic solvent system has
demonstrated high practical potential. The suggested approach allows for the
exclusion of several technological steps during the transformation of
natural b-lactam
antibiotics to their semi-synthetic analogues: alkaline extraction of the
biosynthetic antibiotic from butylacetate followed by its enzymatic hydrolysis
at pH 7.5-8.0 and further acidification of the reaction mixture, which results
in the precipitation of the antibiotic nucleus. Experimental observations also
revealed a specific feature of this process: the kinetic supersaturation of the
antibiotic nucleus slows down the attainment of the equilibrium, which should
be taken into account when further developing this approach.
Halonen P., Baykov A.A., Goldman A., Lahti R., Cooperman B.S.
Biochemistry 41 (2002) 12025-12031
Soluble
inorganic pyrophosphatase (PPase), which converts inorganic pyrophosphate (PPi)
into usable phosphate, is almost universally present as a central enzyme of
phosphorus metabolism and uses divalent metal ion as a necessary cofactor.
PPase from Saccharomyces cerevisiae (Y-PPase) is the best studied with
respect to both structure and mechanism. Here we report the first combined use
of stopped flow and quenched flow techniques to study the PPase reaction in
both the forward (PPi hydrolysis) and back (PPi
synthesis) directions. The results of these studies permit direct comparison of
different divalent metal-ion effects (Mg2+, Mn2+, Co2+)
on microscopic rate constants at pH 7.0. For the Mn-enzyme, on which all of the
high-resolution X-ray studies have been conducted, they demonstrate that the
rate-determining step changes as a function of pH, from hydrolysis of
enzyme-bound PPi at low pH to release of the more tightly bound Pi
at high pH. They also provide evidence for two kinetically important forms of
the product complex EM4(Pi)2, supporting an
earlier suggestion based on crystallographic evidence, and allow informed
speculation as to the identities of acidic and basic groups essential for
optimal PPase catalytic activity.
Komolov K. E., Senin I. I., Filippov P. P.
Russian Journal of Bioorganic Chemistry 28 (2002) 14-19
The role of the
C-terminal domain of rhodopsin in the activation of transducin was studied. The
treatment of photoreceptor membranes with trypsin, thermolysin, and
Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or
19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by
trypsin does not affect the catalytic activity of the receptor, whereas the
thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about
1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19
aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges
catalytic activity of the resulting truncated rhodopsin compared to the
preparation truncated with thermolysin. These results suggest that the part of
the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin
and thermolysin (Val337–Ser338–Lys339)
inhibits the signal transduction from rhodopsin to the next component of visual
cascade.
Kovina M.V., Bykova I.A., Solovjeva O.N., Meshalkina L.E., Kochetov G.A.
Biochemical and Biophysical Research Communications 294 (2002) 155-160
It has long
been known that formation of a catalytically active holotransketolase from the
apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance
of a new band, in both the absorption and CD spectra. Binding and subsequent
conversion of the substrates bring about changes in this band's intensity. The
observation of these changes allows the investigator to monitor the
coenzyme-to-apoenzyme binding and the conversion of substrates during the
transketolase reaction and thus to kinetically characterize its individual
steps. The origin of the thiamin diphosphate induced absorption band has been
postulated to be resulted from formation of a charge transfer complex or
alternatively from an induced conformational transition of the enzyme. The
latter brings aromatic amino acid residues into close proximity and generates
the absorption. However, X-ray crystallographic and enzyme point mutation
experiments cast doubts on both of these hypotheses. Here we show that the
binding of thiamin diphosphate to the apotransketolase leads to the conversion
of the 4'-amino tautomeric form of its aminopyrimidine ring into the
(NH)-H-1'-imino tautomeric form. This imino form emerges as a result of the
coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of
the transketolase active center and is stabilized through the interactions with
Glu418 and Phe445 residues. The N-1'-H-imino tautomeric form of thiamin
diphosphate is thought to be the origin of the holotransketolase absorption
band induced through the coenzyme binding.
van Langen L.M., Janssen M.H.A., Oosthoek N.H.P., Pereira S.R.M., Svedas
V.K., van Rantwijk F., Sheldon R.A.
Biotechnology and Bioengineering 79 (2002) 224-228
Native and
immobilized preparations of penicillin acylase from Escherichia coli and
Alcaligenes faecalis were studied using an active site titration technique.
Knowledge of the number of active sites allowed the calculation of the average
turnover rate of the enzyme in the various preparations and allowed us to
quantify the contribution of irreversible inactivation of the enzyme to the
loss of catalytic activity during the immobilization procedure. In most cases a
loss of active sites as well as a decrease of catalytic activity per active
site (turnover rate) was observed upon immobilization. Immobilization
techniques affected the enzymes differently. The effect of increased loading of
penicillin acylase on the average turnover rate was determined by active site
titration to assess diffusion limitations in the carrier.
Nagradova N.K.
Biochemistry (Moscow) 67 (2002) 839-849
In the process
of oligomeric structure formation through a mechanism of three-dimensional
domain swapping, one domain of a monomeric protein is replaced by the same
domain from an identical monomer. The swapped "domain" can represent
an entire tertiary globular domain or an element of secondary protein
structure, such as an (x-helix or a P-strand. Different examples of
three-dimensional domain swapping are reviewed; the functional importance of
this phenomenon and its role in the development of new properties by some
proteins in the process of evolution are considered. The contribution of three-
dimensional domain swapping to the formation of linear protein polymers and
amyloids is discussed.
Pushkareva M.A., Turutin D.V., Sud'ina G.F.
Cell Biol. Int. 26 (2002) 993-1002
We have
previously demonstrated that the pretreatment of polymorphonuclear leukocytes
(PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment
and inhibits calcium ionophore A23187-stimulated leukotriene (LT) synthesis.
Here, we examined the effects of extracellular arachidonic acid (AA) and
albumin on attachment and LT synthesis in the interaction of PMNs with both
collagen-coated surfaces and human umbilical vein endothelial cell (HUVEC)
monolayers. Suramin decreased the release of radiolabelled AA and
5-lipoxygenase metabolites by [14C-AA]-prelabelled PMNs stimulated
with A23187, with and without human serum albumin (HSA) in the culture medium.
Addition of 1 mM AA together
with calcium ionophore stimulated the release of endogenous AA to the same
level as control and Suramin-pretreated cells, but attachment was unaffected
and LT synthesis was still inhibited with Suramin treatment. Using 24 mM AA, regulation of LT synthesis was
dependent on the presence of HSA in the medium. Without HSA, 24 mM AA induced detachment of PMNs and
increased LT synthesis in Suramin-treated cells above the control level. In the
presence of HSA, 24 mM AA did not influence PMN attachment or abolish Suramin-induced
inhibition of LT synthesis. These results suggest that tight attachment of PMNs
to a solid surface leads to decreased LT synthesis during subsequent
stimulation of the cells by A23187 in the presence or absence of exogenous
substrate.
Roitel O., Ivinova O., Muronetz V., Nagradova N., Branlant G.
Biochemistry 41 (2002) 7556-7564
Tetrameric phosphorylating
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus
stearothermophilus can be described as a dimer of dimers with three
nonequivalent interfaces. To investigate the contribution of intra- and
intersubunit interactions to GAPDH thermostability, 10 residues located either
at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic
domain (amino acids 149-312) were mutated and the thermal unfolding of the
mutants was studied by differential scanning calorimetry in the absence and
presence of saturating concentrations of NAD. Disruptions of intrasubunit
interactions lead to a drastic decrease in thermostability of the N313T, Y283V,
and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative
interactions between the catalytic and the cofactor domains and an inefficient
binding of NAD are observed. This is likely the consequences of modification or
loss of the hydrogen bonding network associating N313 and residues 236-238 and
N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues
Y283 and W310, which are involved in stacking hydrophobic interactions,
mutating both positions does not affect the efficiency of NAD binding. This
shows that the factors involved in the thermostability of the tetrameric apo
GAPDH are then different from those induced by NAD binding. Disruption of
intersubunit hydrogen bonds between the catalytic domain and the NAD-binding
domain of a neighboring subunit also leads to a significant destabilization of
the apo tetrameric form as observed for the D282G mutant. Moreover, no
efficient binding of NAD is observed. Both results are likely the consequence
of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and
R197 and between D282 and R52, respectively. Similar results, i.e., a
destabilizing effect and inefficient NAD binding, are observed with the
T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of
the R-axis-related subunit via mutations at the adenosine subsite. The
dimeric form of the D282G mutant exhibits a single partial heat absorption
peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two
peaks. Taking into account the recent small-angle X-ray scattering studies
which suggested that the dimeric form of the D282G mutant and of the dimeric
Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette,
unpublished results), we propose that the presence of one or two peaks in
thermal unfolding of dimers is a signature of the dimer type.
Salminen A., Parfenyev A.N., Salli K., Efimova I.S.,
Magretova N.N., Goldman A., Baykov A.A., Lahti R.
Journal of Biological Chemistry 277 (2002) 15465-15471
Yeast (Saccharomyces
cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active
sites separated in space from the subunit interface. The present study
addresses the effects of mutation of four amino acid residues at the subunit
interface on dimer stability and catalytic activity. The W52S variant of
Y-PPase is monomeric up to an enzyme concentration of 300 ?M, whereas R51S, H87T, and W279S variants produce
monomer only in dilute solutions at pH greater than or equal to 8.5, as
revealed by sedimentation, gel electrophoresis, and activity measurements.
Monomeric Y-PPase is considerably more sensitive to the SH reagents
N-ethylmaleimide and p- hydroxymercurobenzosulfonate than the dimeric protein.
Additionally, replacement of a single cysteine residue (Cys83),
which is not part of the subunit interface or active site, with Ser resulted in
insensitivity of the monomer to SH reagents and stabilization against
spontaneous inactivation during storage. Active site ligands (Mg2+
cofactor, P-i product, and the PPi analog imidodiphosphate) stabilized the
W279S dimer versus monomer predominantly by decreasing the rate of dimer to
monomer conversion. The monomeric protein exhibited a markedly increased
(5-9-fold) Michaelis constant, whereas kcat remained virtually
unchanged, compared with dimer. These results indicate that dimerization of
Y-PPase improves its substrate binding performance and, conversely, that active
site adjustment through cofactor, product, or substrate binding strengthens
intersubunit interactions. Both effects appear to be mediated by a
conformational change involving the C-terminal segment that generally shields
the Cys83 residue in the dimer.
Shchepina N.E., Nefedov V.D., Toropova M.A., Badun
G.A., Avrorin V.V., Fedoseev V.M.
Radiochemistry 44 (2002) 378-379
Nuclear-chemical
synthesis of halonium compounds was studied, and the influence exerted by the
structure of substituent [R = H, 4-Br, 4-CH3, 2-CH3,
2,4,6-(CH3)3, 4-NO2, 2,3,4,5,6-F] in
substituted halobenzene substrate molecule on the yield of the onium compound
was examined. Electron-withdrawing (bromo, nitro, perfluoro) and sterically
shielding (2,4,6-trimethyl) substituents fully suppress formation of the
corresponding onium compound. At the same time, the electron-donor methyl group
in the p-position of the ring (s = -0.17) activates the lone electron pair of the halogen, which
increases the yield of the onium compound. This is especially important in the
case of unknown diarylfluoronium derivatives which can be prepared today by a
nuclear-chemical procedure only.
Arachidonic acid
and docosahexaenoic acid suppress thrombin- evoked Ca2+ response in rat astrocytes by endogenous arachidonic acid
liberation.
Sergeeva M., Strokin M., Wang H., Ubl J.J., Reiser G.
Journal of Neurochemistry 82 (2002) 1252-1261
Arachidonic
(AA) and docosahexaenoic acid (DHA) are the major polyunsaturated fatty acids
(PUFAs) in the brain. However, their influence on intracellular Ca2+
signalling is still widely unknown. In astrocytes, the amplitude of thrombin-
induced Ca2+ response was time-dependently diminished by AA and DHA,
or by the AA tetraynoic analogue ETYA, but not by eicosapentaenoic acid (EPA).
Thrombin-elicited Ca2+ response was reduced (20-30%) by 1-min
exposure to AA or DHA. Additionally, 1-min application of AA or DHA together
with thrombin in Ca2+-free medium blocked Ca2+ influx,
which followed after readdition of extracellular Ca2+. EPA and ETYA,
however, were ineffective. Long-term treatment of astrocytes with AA and DHA,
but not EPA reduced the amplitude of the thrombin-induced Ca2+
response by up to 80%. AA and DHA caused a comparable decrease in intracellular
Ca2+ store content. Only DHA and AA, but not EPA or ETYA, caused
liberation of endogenous AA by cytosolic phospholipase A2 (cPLA2). Therefore,
we reasoned that the suppression of Ca2+ response to thrombin by AA
and DHA could be due to release of endogenous AA. Possible participation of AA
metabolites, however, was excluded by the finding that specific inhibitors of
the different oxidative metabolic pathways of AA were not able to abrogate the
inhibitory AA effect. In addition, thrombin evoked AA release via
activation of cPLA2. From our data we propose a novel model of
positive/negative-feed-back in which agonist- induced release of AA from
membrane phospholipids promotes further AA release and then suppresses
agonist-induced Ca2+ responses.
Shishkov A.V., Ksenofontov A.L., Bogacheva E.N.,
Kordyukova L.V., Badun G.A., Alekseevsky A.V., Tsetlin V.I., Baratova L.A.
Bioelectrochemistry 56 (2002) 147-149
The topography
of bacteriorhodopsin (bR) in situ was earlier studied by using the
tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the
X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95
(1998) 11673], we estimated the influence of membrane environment (lipid and
protein) on tritium incorporation into amino acid residues forming
transmembrane helices. We have determined the tritium flux attenuation
coefficients for residues 10-29 of helix A. They turned out to be low
(0.04 ± 0.02 A-1))
for residues adjacent to the lipid matrix, and almost fourfold higher
(0.15 ± 0.05 A-1)
for those oriented to the neighboring transmembrane helices. We believe that
tritium incorporation data could help modeling transmembrane segment
arrangement in the membrane.
Isolation and
properties of noncovalent complex of transketolase with RNA
Solovjeva O.N.
Biochemistry (Moscow) 67 (2002) 667-
A method for
isolation of homogenous transketolase from baker's yeast using immunoaffinity
chromatography was significantly simplified. It was demonstrated that
transketolase could be isolated from fresh yeast in the form of a complex with
a high molecular weight RNA. Storage of yeast led to the dissociation of the
complex to a low molecular weight complex and then to the free enzyme.
Conditions were chosen for complex dissociation and free enzyme isolation. In
comparison to the free enzyme, the specific activities of the high and low
molecular weight complexes were decreased 20-25- and 3-5.5- fold, respectively
The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate
(donor substrate) did not change for the low molecular weight complex, while
the time of binding to calcium increased. The latter was necessary for the
complete manifestation of the enzymatic activity. Changes in the circular
dichroism spectrum between 300 and 360 nm after the addition of thiamine
diphosphate, which characterize the formation of the catalytically active
holoenzyme, were significantly lower for the low molecular weight complex than
for the free enzyme.
Ubl J.J., Grishina Z.V., Sukhomlin T.K., Welte T.,
Sedehizade F., Reiser G.
American Journal of Physiology - Lung Cellular and
Molecular Physiology 282 (2002) L1339-L1348
Protease-activated
receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology.
Proteases cleaving the extracellular NH2 terminus of receptors activate or
inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and
immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human
bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was
confirmed by Ca2+ imaging studies using the receptor agonist
protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked
by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of
PAR-2-elicited Ca2+ response in HBE and A549 cells depend on
concentration and time of agonist superfusion. The response is partially
pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor
U- 73122, and diminished by the inositol 1,4,5-trisphosphate receptor
antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the
resting Ca2+ level nor PAR-2-elicited Ca2+ response.
Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent
Ca2+ response in HBE, but not A549, cells. In both cell lines,
thermolysin abolished the response to a subsequent trypsin challenge but not to
SLIGKV. Thus different epithelial cell types express different PAR-2 with
identical responses to physiological stimuli (trypsin, SLIGKV) but different
sensitivity to modifying proteases, such as thermolysin.
Hexameric, Trimeric, Dimeric,
and Monomeric Forms of Inorganic Pyrophosphatase from Escherichia coli
Vainonen Yu.P., Kurilova S.A., Avaeva S.M.
Russian Journal of Bioorganic Chemistry 28 (2002) 385-391
The conditions
were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia
coli inorganic pyrophosphatase from its native hexameric form.
Interconversions of the oligomers were studied, and rate constants for their
dissociation and association were determined. All forms were found to be
catalytically active, with the activity decreasing in the following order:
hexamer–trimer–dimer–monomer. The activity of trimeric and dimeric forms was
high enough to study and to compare their catalytic properties. The monomeric
form of the enzyme was unstable.
Voronov S., Zueva N., Orlov V., Arutyunyan A., Kost O.
FEBS Letters 522 (2002) 77-82
Somatic
angiotensin-converting enzyme (ACE) consists of two homologous domains, each
domain bearing a catalytic site. Differential scanning calorimetry of the
enzyme revealed two distinct thermal transitions with melting points at 55.3
and 70.50C. which corresponded to denaturation of C- and N- domains,
respectively. Different heat stability of the domains underlies the methods of
acquiring either single active N- domain or active N-domain with inactive
C-domain within parent somatic ACE. Selective denaturation of C-domain supports
the hypothesis of independent folding of the two domains within the ACE
molecule. Modeling of ACE secondary structure revealed the difference in
predicted structures of the two domains, which, in turn, allowed suggestion of
the region 29133 in amino acid sequence of the N-part of the molecule as
responsible for thermostability of the N-domain.
Youshko M.I., Chilov G.G., Shcherbakova T.A., Svedas V.K.
Biochimica et Biophysica Acta - Proteins Proteomics 1599 (2002) 134-140
Nucleophile
reactivity of two most known nuclei of penicillins and cephalosporins,
6-aminopenicillanie (6-APA) and 7- aminodesacetoxycephalosporanic (7-ADCA)
acids, was quantitatively characterized. In penicillin acylase (PA)- catalyzed
acyl transfer reactions the relative reactivity of the added nucleophile
compared to the water (i.e. nucleophile reactivity) is defined by two complex
kinetic parameters b(0) and g, and depends
on the nucleophile concentration. In turn, parameters b(0) and g were shown to be dependent on the
structure of both reactants involved: nucleophile and acyl donor. Analysis of
the kinetic scheme revealed that nucleophile reactivity is one of a few key
parameters controlling efficiency of PA-catalyzed acyl transfer to the added
nucleophile in an aqueous medium, Computation of the maximum nucleophile
conversion to the product using determined nucleophile reactivity parameters in
the synthesis of three different antibiotics, ampicillin, amoxicillin and
cephalexin, showed good correlation with the results of corresponding synthetic
experiments, Suggested approach can be extended to the quantitative description
and optimization of PA-catalyzed acyl transfer reactions in a wide range of
experimental conditions.
Youshko M.I., van Langen L.M., de Vroom E., van Rantwijk F., Sheldon
R.A., Svedas V.K.
Biotechnology and Bioengineering 78 (2002) 589-593
The penicillin
acylase-catalyzed synthesis of ampicillin by acyl transfer from
D-H-phenylglycine amide (D-PGA) to 6- aminopenicillanic acid (6-APA) becomes
more effective when a judiciously chosen pH gradient is applied in the course
of the process. This reaction concept is based on two experimental
observations: 1) The ratio of the initial synthesis and hydrolysis rates (VsNH)
is pH-dependent and exhibits a maximum at pH 6.5-7.0 for a saturated solution
of 6APA; 2) at a fixed 6-APA concentration below saturation, VsNH increases
with decreasing pH. Optimum synthetic efficiency could, therefore, be achieved
by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing
the pH to 6.3 in the course of 6- APA consumption. A conversion of 96% of 6-APA
and 71% of DPGA into ampicillin was accomplished in an opitimized procedure,
which significantly exceeds the efficiency of enzymatic synthesis performed at
a constant pH of either 7.0 or 6.3.
Zyryanov A.B., Baykov A.A.
Biochemistry (Moscow) 67 (2002) 635-639
Complex
formation between Arsenazo III and Mn2+ and Co2+ at
equilibrium has been investigated at PH 7.2, and the stoichiometry and
stability of the complexes have been determined. The data indicate that
Arsenazo III is suitable for determination of Mn2+ and Co2+
on the mmolar scale.
The dissociation constants of the phosphate complexes of Mn2+ and Co2+
at PH 7.2 were estimated with Arsenazo III as 3.6 and 10 mM, respectively.