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ORIGINAL ARTICLE
Lactobacilli and bifidobacteria lectins as possible signal molecules
regulating intra- and inter-population bacteria
bacteria and
host
bacteria relationships. Part I. Methods of bacterial lectin
isolation, physico-chemical characterization and some biological
activity investigation
VLADIMIR M. LAKHTIN, MICHAIL V. LAKHTIN, VERONIKA V. POSPELOVA &
BORIS A. SHENDEROV
Gabrichevsky Epidemiology and Microbiology Research Institute, Moscow, Russia
Abstract
Lectins (natural or artificial nonimmunoglobulin origin proteins/glycoproteins) are able to bind noncovalently with
carbohydrate targets. Lectins and lectin-like substances of lactobacilli (L. acidophilus K
3
III
24
; 100ash; NK1; L. plantarum
8RA3) and bifidobacteria (B. adolescentis MC42; B gallinarum, B. bifidum 1) have been investigated. Bacteria were grown in
semi-anaerobic conditions in a casein-yeast broth. Medium was microfiltrated and ultrafiltrated for isolation of 30 kDa
compounds which were then separated by isoelectric focusing in polyacrylamide gel in the presence of urea and sucrose.
Components isolated were electrotransferred to immobillon P. Blots obtained were then treated with a set of biotinylated
artificial linear homopolysaccharides revealed by streptavidin-peroxidase conjugate. Chemiluminescence registration of
peroxidase bound to blots was performed using the ‘BioChemi System’ camera. A spectrum of different polysaccharide-
binding individual components reacting with such homopolymers as mannan-alpha (phosphorylated or not), GalNAc-
alpha-polysaccharide of mucin-like-type or galactan-beta (sulfated or not)-binding was found in lactobacilli and
bifidobacteria. Lactobacilli produced a more limited spectrum of lectins or lectin-like compounds with higher affinities
to polysaccharides tested than bifidobacteria having more extended pI polysaccharide-binding activity spectrum. Interaction
of isolated lectins with T and B lymphocytes, macrophages, and thrombocytes was demonstrated. The importance of
lactobacilli and bifidobacteria lectins/lectin-like components and their effector complexes in cell mitoses, blood coagulation,
aerobic metabolic reactions, and other biological activities is discussed.
Key words: bacterial lectin, lactobacilli, bifidobacteria, bacterial communication, bacteria host cross-talk, lectin isolation,
lectin identification, lectin biological activity
Introduction
There is great interest in the processes of intra-
and inter-population communication of bacteria
with each other (quorum sensing) and bacteria
with their host cells (cross-talk). These processes
are known to be involved in regulation of bacterial
colonization, biofilm formation, antibiotic and
bacteriocin production, pathogenicity characteris-
tics (activity and expression of many virulence
factors), colonization resistance, etc. in numerous
bacterial systems and bacteria host systems (13).
Within such relationships, it is particularly impor-
tant to understand a phenomenon of communica-
tion between lactobacilli and bifidobacteria and the
host organism. These representatives of the normal
intestinal microflora actively participate in coloni-
zation resistance, immune system regulation, and
other physiological and metabolic host functions
(46). Many bacteria bacteria and bacteria host
communication regulation system signals have
been isolated and investigated. The best known
among them are nitrogen oxide, hydrogen perox-
ide, some lactones, peptides, amines, short-chain
fatty acids, oligosaccharides, amino acids, and
lectins (13,7).
Correspondence: Professor Boris A. Shenderov, MD, Gabrichevsky Epidemiology and Microbiology Research Institute, Admirala Makarova Street 10,
Moscow 125212, Russia. Tel:
/7 095 452 18 16. Fax: /7 095 452 18 30. E-mail: info@gabrich.com; shenderov@starnet.ru
Microbial Ecology in Health and Disease. 2006; 18: 55 60
(Received 14 December 2005; accepted 8 May 2006)
ISSN 0891-060X print/ISSN 1651-2235 online #2006 Taylor & Francis
DOI: 10.1080/08910600600799646
Among signal molecules regulating the interac-
tions noted above, an important role for lectins (the
family of galectins, and others) in different types
of communications (bacteria bacteria, biofilm for-
mation, bacterium cell, cell tissue organwhole
organism, cellcell organelles, etc.) is postulated
(711).
Some recent publications have dealt with the
isolation and investigation of different lectins (12
14), but lectins of bifidobacteria and lactobacilli have
not yet been seriously investigated, and few data are
available in the literature (1517).
The aim of the present study was to develop
simple and reproducible methods of isolation, iden-
tification and investigation of lactobacilli and bifido-
bacteria lectins and lectin-like compounds.
Materials and methods
Microorganisms
Seven strains representing three species of bifido-
bacteria (B. adolescentis MC 42, B. bifidum 1 and
B.gallinarum ) and four strains of lactobacilli (L.
acidophilus 100ash, NK1, K
3
III
24
;L. plantarum
8RA3) were tested. The strain of B. gallinarum
(given by G.I. Bovkun, Bryansk Veterinary Acad-
emy, Russia) was isolated from faeces of healthy
fowl. All other strains were of human origin and are
used as probiotic starter cultures in Russia (obtained
from Gabrichevsky Institute Collection, Moscow).
Medium
‘Bifidum medium’ (Biomed, Moscow oblast, Russia)
was used for cultivation of lactobacilli and bifido-
bacteria. It consists of (g/L): yeast extract, 5.0;
pancreatic casein hydrolysate, 30.0; glucose, 7.5;
lactose, 2.5; cysteine, 0.5; NaCl, 2.5; MgSO
4
, 0.5;
CH
3
COOHNa, 0.3; ascorbic acid, 0.5; agar, 0.75.
The pH of the medium was 6.87.0.
Reagents
Buffers. These comprised 10 mM Veronal-medinal
saline buffer (VBS), pH 7.4, with 0.85% NaCl, 0.5
mM MgCl
2
, 0,15 mM CaC1
2
(VBS
2
); and 10 mM
phosphate saline buffer, pH 7.4 (PBS), which was
prepared from tablets (Bio-Rad Lab, USA).
Reagents for isoelectric focusing (IEF) in polyacrylamide
gel (IEF-PAAG) and blotting. The reagents were as
follows. A 40% mixture of acrylamide-bisacrylamide
(Bio-Rad); ampholines Bio-Lyte 3 5 (Bio-Rad),
urea (Ultragrade, Hercules, USA), sucrose (extra;
Helicon, Moscow), ammonium persulphate (ACS
Grade; Helicon), TEMED (ultra pure grade; Sigma,
USA), methyl red with pI 3.75 (Sigma), Tris (ultra
pure grade; Amresco, USA), Gly (biotechnology
grade; Helicon), a cocktail of proteinase inhibitors
‘‘Complete’’ (tablets, Roche, Germany), Na
2
-EDTA
( Sigma), dithiotreitol (Merck, Germany). Tween-80
(Aldrich Chemical Company, USA), streptavidin-
peroxidase (R&D Systems, Minneapolis, USA),
conjugate of streptavidin-peroxidase (Streptavidin-
HRP; R&D Systems); extended duration chemilu-
minescent substrate BioWest (UVP Bioimaging
Systems, CA, USA); water of Milli Quality (Milli-
pore System, USA) were also used. Artificial linear
soluble homopolysaccharides (ALSHP) in biotiny-
lated or nonbiotinylated form (Syntesome, GmbH,
Moscow; Russia). Free polysaccharide probes were a
kind gift from Professor N.V. Bovin (Inst. Bioorganic
Chemistry, Moscow), lyophilized in glass vials (1
mg), having 20% glycoside covalently linked to poly-
acrylamide (PAA) (GalNAc-alpha-PAA; Gal-beta-
PAA; Gal-sulphate-beta-PAA; Man-alpha-PAA;
Man-6-phosphate-alpha-PAA; GlcNAc-beta-PAA).
Materials for IEF-PAAG
Hydrophilic Durapore membrane (DVPP, 0.65 mm
pores, Millipore, USA), hydrophobic immobillon-P
membrane (PVDF, 0.45 mm pores, Millipore), and
Gelbond film as support of gel (Amersham Pharma-
cia Biotech AB, Sweden) were utilized in the study.
Equipment
The equipment comprised High Voltage Power
Supply (Amersham Pharmacia Biotech, Sweden);
Multiphore II (Amersham Pharmacia Biotech);
Multitemp (Amersham Pharmacia Biotech); Nova
blot for combination with Multiphore II; and Epi
Chemi II Darkroom as a part of the BioChemi
System (UVP Bioimaging Systems, CA, IIIA).
The program LabWorks Version 4.0 (UVP Bioi-
maging Systems) was used.
Approaches
Because the affinity of the majority of known lectins
is relatively low and in addition, admixtural hydro-
phobic proteins (such as bovine serum albumin used
for the blot blocking) can interfere with some lectins,
a set of approaches for the isolation and study of
multiple probiotic bacterial lectin forms has been
developed by the authors.
To study all possible forms of bacterial lectins, we
used blotting analysis to identify agglutinating and
nonagglutinating (monovalent) forms of lectins;
improved the sensitivity of the cytoagglutination
test using lectin sorption on the bottom of the
56 V.M. Lakhtin et al.
microplate wells as well as on the cells in the same
wells; excluded protein (BSA) on the blot membrane
(PVDF); used artificial soluble homopolysacchar-
ides (ALSHP) as the most sensitive lectin probes
with known structures; used a biotin-streptavidin
system as more sensitive one to detect biotinylated
polysaccharide binding with peroxidase conjugate;
used a very sensitive kinetic chemilumescent detec-
tion of bound peroxidase; used a sensitive fluores-
cent stain to visualize protein bands in tracks;
registered fluorescence and chemiluminescence as a
set of sequential kinetic files (TIFF) of live images to
be edited, calculated and/or compared.
Procedures for isolation of lactobacilli or bifidobacteria
lectins/adhesins
Procedure I. 1) Bacterial growth in culture. 2) Sample
preparation: a) culture centrifugation (supernatant);
b) microfiltration and sterilization using Steriflip
with Millipore Express Membrane GP, 0.22 mm
pores (Millipore) and vacuum pump (Millipore)
(filtrate); c) concentration and concentrate washing
in the presence of protease inhibitor cocktail ‘Com-
plete’ (Roche) using ultrafiltration centrifugation
through Centricon Plus20 (Millipore) (bacterial
components having a molecular mass of at least
30 kDa); d) thermal treatment of the concentrates
followed by addition of Tween-80 into concentrates
(before IEF-PAAG); e) separation of bacterial com-
ponents in a gel block (11
/25/0.12 cm) by
horizontal IEF (pH 4 8)-PAAG (5%)-urea (8 M)-
sucrose (5%) in pH gradient 4 8 (Servalyt 4 6,
Serva, Germany; Bio-Lyte 6 8, Bio-Rad) at 108C,
according to Lasne et al. (19) for glycoproteins (with
modification), using Multiphore II (Pharmacia Bio-
tech) and High Voltage Power Supply (Amersham
Pharmacia Biotech); f) extraction of lectins from the
known position in gel tracks (preliminary lectin
testing is needed) and their concentration as for
concentrate (see above ‘c’).
Procedure II (for lactobacilli cell surface lectins/adhesins)
according to Kang et al. (20), with modifications. 1)
Bacterial growth in culture. 2) Sample preparation:
a) culture centrifugation (cells); b) few washings of
the cell pellet with H
2
O; c) sequential treatments of
cells with 0.2 M NaCl and urea; d) extraction of cell
adhesins/lectins with 3 M LiCl; e) thermal treatment
of cell residue by mixture of 1% DDS-Na and 0.5%
2-mercaptoethanol; f) precipitation of urea super-
natant with acetone followed by dissolution of
precipitate; 3) IEF of samples (before ‘d’, ‘e’, ‘f ’,
and after ‘f’) and identification of lectins.
Procedure III (for lactobacilli lectins/adhesins from
cultural fluid). 1) Obtain bacterial components (see
Procedure I). 2) Sample preparation: a) acetone
treatment of retentates to eliminate insoluble dark
gum-like material; b) dissolution of protein precipi-
tates (not gum-like) in the presence of protease
inhibitors. 3) IEF and identification of lectins.
Protein in purified lectin samples was measured
according to the Waddell method (18). IEF in a
horizontal plate (24
/12/0.12 cm) of 5% PAAG in
the presence of 8 M urea and 5% sucrose, and
blotting were performed according to the method for
separation of hydrophobic and glycosylated compo-
nents of biological fluids described by Lasne et al.
(19), with modification. pH gradients used were 2 6
or 48. Electrolytes used were 0.1 M NaOH as
cathode and 10 mM H
3
PO
4
as anode.
Lectin testing on blots
The testing was carried out as follows. 1) Semi-dry
electrotransfer (blotting) from IEF-PAAG on sand-
wich including hydrophilic Durapore membrane
(DVPP, 0.65 mm pores, Millipore) and hydrophobic
immobillon-P membrane (PVDF, 0.45 mm pores,
Millipore) using Nova blot graphite plate electrodes
(in combination with Multiphore II). 2) Fluorescent
visualization for one part of the blot using SYPRO
Ruby protein blot stain (Bio-Rad). 3) Chemilumi-
nescent lectin-specific visualization for another sym-
metrical part of the blot using a set of biotinylated
ALSHP as polyacrylamide (PAA) chains having
side glycosides (Syntesome GmbH). The blot was
treated with the freshly made biotinylated polysac-
charide probe (5 20 mg/ml in PBS with 0.01%
Tween-80) overnight at 48C followed by treatment
with streptavidin-peroxidase (1:2000, v/v) in PBS
with 0.01% (v/v) Tween-80 for 1 h at 378C. The
bound peroxidase was detected with BioWest (UVP
Bioimaging Systems). 4) Registration of fluorescent
and chemiluminescent pictures in the Camera Epi
Chemi II Darkroom of the BioChemi System (UVP
Bioimaging Systems) using the programme Lab-
Works Version 4.0.
Cytoagglutination monitoring of bacter ial lectins
This was performed in U-bottomed polystyrene 96-
well plates (MedPolymer, Moscow) in serial dilu-
tions of sample in 10 mM PBS, pH 7.4, or 10 mM
veronal-medinal saline buffer with Ca
2
and Mg
2
(VBS
), pH 7.4. Test systems were fresh and
extensively washed with PBS human erythrocytes
AII(
/) from a healthy donor; erythrocytes were
treated with N-acetylneuraminidase from Clostri-
dium perfringens (type VIII, Sigma) or trypsin
Identification, isolation and biological activities of lactobacilli and bifidobacteria lectins 57
(Sigma); cells of commercial lyophilized (S.I.
Lesaffe, France) or fresh baker’s yeast Saccharomyces
cerevisiae . Evaluation of the final cytoagglutinating
pictures was performed in the camera of the Bio-
Chemi System. Inhibition of purified lactobacilli and
bifidobacteria lectins with ALSHP was tested by
addition of ALSHP (5 20 mg/ml) to the wells that
had cytoagglutinates followed by resuspension of the
cells in wells. As a result agglutinates were comple-
tely or partially eliminated.
Investigation of lectins as autoregulators
This procedure was developed by the authors. This
part of the work was performed in a minivolume
reactor such as a 1 ml volume insulin sterile syringe:
550 ml of the purified protein endogenous auto-
regulator
/0.1 ml of probiotic lactobacilli or bifido-
bacteria concentrate
/0.9 ml of growth medium for
lactobacilli and bifidobacteria. Results were moni-
tored for 1, 2, 3 and more days after incubation at
378C in anaerobic condtions (for bifidobacteria) or
partially aerobic conditions at the top of syringes (for
lactobacilli). The criteria for the autoregulator influ-
ence for lactobacilli were change of color of medium
(in anaerobic conditions) and/or decreasing turbidity
at the top (in aerobic conditions). Criteria for
bifidobacterial autoregulator action in the optically
transparent medium were change of color and
increasing turbidity of medium; number, shape and
size of the bifidobacterial colonies, character of
colony distribution and adhesion to the walls along
the plastic syringe (top-middle-bottom). These de-
tails of the bifidobacterial autoregulators’ actions
were registered with the naked eye and/or in the
camera of the BioChemi System.
Results and discussion
Isolation and characterization of bifidobacteria and
lactobacilli lectins
There are many examples of lectin-like interactions
of lactobacilli or bifidobacteria strains with a panel of
simple sugars, oligosaccharides or polysaccharides,
glycoproteins, and/or glycolipids (12). The data
connected with lactobacilli purified lectin-like pro-
teins have been very limited (15 17). In the case of
bifidobacteria they are completely absent. There are
no publications about the lectins isolated from
probiotic lactobacilli and bifidobacteria cultural
fluids at all. The new approaches and procedures
developed by the authors made it possible to obtain
and identify a panel of lectins with exact carbohy-
drate specificities from probiotic lactobacilli and
bifidobacteria strains separated from colored acid
admixtures (pIB
/3.0), polysaccharides, and nucleic
acids.
L. acidophilus purified lectins (procedure I) were
isolated. Acidic lectins (pI 3.5 4) and basic lectins
(pI around 8) of lactobacilli agglutinated neurami-
nidase-treated erythrocytes with titers of 5.4 and 2.1
mg/ml, respectively. In the reaction with neuramini-
dase-treated erythrocytes, the purified lectins from
L. acidophilus were inhibited by (GalNAc-alpha)
n
-
PAA and, to a lesser extent, by (Man-alpha)
n
-PAA
(for acidic lectins), and by (GalNAc-alpha)
n
-PAA
(for basic lectins).
Bifidobacteria lectins (procedure I) were isolated.
B. gallinarum acidic lectins (pI 3.5 4) and basic
lectins (pI around 8) agglutinated neuraminidase-
treated erythrocytes with titers of 1.3 and 6.0 mg/ml,
respectively.
In the reaction with neuraminidase-treated ery-
throcytes, acidic and basic purified lectins from B.
gallinarum ,B. adolescentis MC42, and B. bifidum 1
were inhibited by (GalNAc-alpha)
n
-PAA and, to a
lesser extent, by (Man-alpha)
n
-PAA.
In the case of IEF in a more acidic gradient, pH
26, lactobacilli (L. acidophilus and L. plantar um )
produced a more limited spectrum of lectins or
lectin-like compounds with higher affinities to (Man-
alpha)
n
-PAA than bifidobacteria (B. adolescentis
MC42). L. plantarum produced more (Man-al-
pha)
n
-PAA-specific lectins than L. acidophilus.
(Man-alpha)
n
-PAA resulted in higher intensive
chemiluminescence than (Gal-beta)
n
-PAA, and
(GlcNAc-beta)
n
-PAA did not bind to the blots
at all. Carbohydrate specificities of lactobacilli and
bifidobacteria were correlated with their coag-
glutination activity towards yeast cells, and neu-
raminidase-treated or trypsinized human A(II)
erythrocytes.
Lactobacilli LiCl-extracted surface lectins (proce-
dure II) and acetone-extracted lectins from cultural
fluids (procedure III) were isolated. They had
purified proteins spectral characteristics. The pur-
ified lactobacilli LiCl-extracted lectin agglutinated
neuraminidase-treated erythrocytes at concentra-
tions of 0.5 mg/ml (the titer) and higher. This
agglutination was inhibited with (GalNAc)
n
-PAA,
but not with (Man)
n
-PAA. Acetone-extracted lectins
from cultural fluids (procedure III) agglutinated
neuraminidase-treated erythrocytes at a relatively
low titer
/142 mg/ml, and this agglutination was
inhibited with (GalNAc)
n
-PAA and, to a lesser
extent, with (Man)
n
-PAA.
Cytoagglutination of lactobacilli and bifidobac-
teria lectins was Ca
2
- and Mg
2
-independent.
The extended panel of lectins (obtained according
to procedures I, II, and III) separated in the gradient
58 V.M. Lakhtin et al.
pH 48 reacted with ALSHP tested (Tables I and
II).
Representatives of all species of bifidobacteria
tested (Table I) had a unique individual spectrum
of lectins; B. adolescentis produced maximal levels of
lectins (maximal intensities). All lactobacilli strains
investigated also possessed an individual spectrum of
lectins (Table II).
Taking into consideration a potentially high hy-
drophobicity of bacterial lectins, we studied carbo-
hydrate specificity of lectins at the level of their
reduced polypeptide forms treated with Na-dode-
cylsulphate and beta-mercaptoethanol (Table II). As
a result, more multiple individual forms of lactoba-
cilli lectins could be evaluated, and higher intensities
(affinities) could be registered.
Thus, using a number of procedures elaborated by
the authors of the present study, a wide spectrum of
different polysaccharide-binding individual lectins in
probiotic lactobacilli and bifidobacteria strains was
isolated and identified. These lectins were able to
recognize mannan-alpha (phosphorylated or not)
homopolymers (analogs of the yeast mannans);
N-acetylgalactosamine-a-homopolymer (analog of
the exposed intestinal mucin carbohydrate moiety);
or galactose-homopolymer (sulfated or not) (the
other analog of exposed mucus).
Biological activities of Lactobacillus and Bifidobacterium
lectins
The cytoagglutinating activity of Lactobacillus or
Bifidobacterium lectins decreased with respect to the
following cell test systems: desialylated human ery-
throcytes AII(
/)/trypsinized human erythrocytes
AII(
/)/yeast cells//native human erythrocytes.
As regards autoregulating activity, the use of
minibioreactors is very important in the study of
mammalian cells, but examples are not available for
probiotic strains of lactobacilli and bifidobacteria
(21).
As regards autoregulating activity in the system ‘B.
gallinarum -secreted purified proteins of B. galli-
narum ’ (anaerobic conditions), our semi-anaerobic
minibioreactor was especially useful in monitoring of
colonial growth of bifidobacteria. Endogenic pro-
teins stimulated biomass (size) of colonies as well as
increased number of colonies (mitogen-like action).
In the case of autoregulating activity in the system
‘L. acidophilus as a mixture of three strainssecreted
Table I. Specificity of multiple forms of Bifidobacterium lectins pu-
rified and separated in 8 M urea from complexes with molecular
masses of at least 30 kDa.
Strain Specificity pI* Intensity$
B. adolescentis MC42 GalNAc-PAA 7.5 84
//3/
Man-6P-PAA 6; 8 3///
Gal-SU-PAA 45; 7 4///
B. gallinarum GalNAc-PAA 8 2/
Man-6P-PAA 6.5; 7.5 82////3/
Gal-SU-PAA 7 2/
B. bifidum 1 GalNAc-PAA 7 7.5 2/
Man-6P-PAA 8 2/
Gal-SU-PAA 4 54/
*Calculated from the linear gradient of pH created.
$Where 4/was the maximal intensity of the chemiluminescence.
Table II. Specificity of multiple forms of Lactobacillus lectins purified and separated in 8 M urea from complexes with molecular masses of
at least 30 kDa.
Strain Specificity pI* Intensity$
LiCl-extracted lectins
L. acidophilus (mixture of 100ash, NK1, K
3
III
24
) GalNAc-PAA 8 /
6/
Gal-SU-PAA 5 /
L. acidophilus 100ash GalNAc-PAA 5.1 77.5; 8 //4//3/
Ma n-6P-PAA 6.5 2/
Gal-SU-PAA 5; 7 //3/
L. acidophilus NK1 GalNAc-PAA 8 2/
Man-6P-PAA 6; 6.5; 7 //2///
(DDS/ME) extracted lectins
L. acidophilus (mixture of 100ash, NK1, K
3
III
24
) GalNAc-PAA 4.55.5; 5.8; 6.3 3//3/2/
Man-6P-PAA 5.5 6.5 2//3//2/
L. acidophilus 100ash GalNAc-PAA 5.8; 6.2 ///
Ma n-6P-PAA 6 72///
Gal-SU-PAA 5; 7 //3/
L. acidophilus NK1 GalNAc-PAA 4.5 5; 5.7; 6.2 3//2///
Man-6P-PAA 5; 6 6.8; 7 82//2//4/
DDS, dodecyl sulfate; ME, mercaptoethanol.
*Calculated from the linear gradient of pH created.
$Where 4
/was the maximal intensity of the chemiluminescence.
Identification, isolation and biological activities of lactobacilli and bifidobacteria lectins 59
purified endogenic proteins’, lectin fraction from the
‘urea-treated step’ and, to a lesser extent, from
‘acetone treatment step’ stimulated aerobic metabo-
lism of lactobacilli in a dose-dependent manner in 3
days of the growth.
Perspectives
At present, the work includes the following main
directions. 1) Large-scale isolation of a set of
probiotic bacterial lectins. 2) Study of their biologi-
cal and physiological activities at the level of the
mammalian host organism and its organs, tissues,
and cells. Study of the interaction of isolated lectins
with T and B lymphocytes, macrophages, and
thrombocytes is in progress. 3) Study of the role of
lactobacilli and bifidobacteria lectins in a mixture of
microbial cultures and artificial biofilms. 4) Proteo-
mic analysis of lectins for creation of artificial lectins
with new useful physico-chemical and biological
characteristics.
Acknowledgements
The authors wish to thank Professor N.V. Bovin
(Inst. Bioorganic Chemistry, Moscow) for the poly-
saccharide probes.
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