Application of Peak Intensity Analysis to Measurements of Protein Binding to Lipid Vesicles and Erythrocytes Using Fluorescence Correlation Spectroscopy: Dependence on Particle Size.

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool for investigation of processes accompanied by changes in the mobility of molecules and complexes. In the present work, peak intensity analysis (PIA) in combination with the solution stirring using FCS setup was applied to explore the interaction between fluorescently labeled protein ligands and corresponding receptors located on membranes. In the system composed of biotinylated liposomes and fluorescently labeled streptavidin as a ligand, PIA allowed us to determine the optimum receptor concentration and demonstrate the essential dependence of the binding efficacy on the length of the linker between the biotin group and the polar head group of the lipid. The binding was dependent on the size of liposomes which was varied by lipid extrusion through filters of different pore diameters. The sensitivity of the method was higher with the liposomes of larger sizes. The PIA approach can be applied not only to liposomes but also to relatively large objects, e.g., erythrocytes or Sepharose beads derivatized with lactose as a receptor for the binding of viscumin and ricin.

Photodynamic activity of the boronated chlorin e6 amide in artificial and cellular membranes.

Photodynamic tumor-destroying activity of the boronated chlorin e6 derivative BACE (chlorin e6 13(1)-N-{2-[N-(1-carba-closo-dodecaboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester), previously described in Moisenovich et al. (2010) PLoS ONE 5(9) e12717, was shown here to be enormously higher than that of unsubstituted chlorin e6, being supported by the data on much higher photocytotoxicity of BACE in M-1 sarcoma cell culture. To validate membrane damaging effect as the basis of the enhanced tumoricidal activity, BACE was compared with unsubstituted chlorin e6 in the potency to photosensitize dye leakage from liposomes, transbilayer lipid flip-flop, inactivation of gramicidin A ionic channels in planar lipid membranes and erythrocyte hemolysis. In all the models comprising artificial and cellular membranes, the photodynamic effect of BACE exceeded that of chlorin e6. BACE substantially differed from chlorin e6 in the affinity to liposomes and erythrocytes, as monitored by fluorescence spectroscopy, flow cytometry and centrifugation. The results support the key role of membrane binding in the photodynamic effect of the boronated chlorin e6 amide.

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds.

Bioengineered scaffolds are crucial components in artificial tissue construction. In general, these scaffolds provide inert three-dimensional (3D) surfaces supporting cell growth. However, some scaffolds can affect the phenotype of cultured cells, especially, adherent stromal cells, such as fibroblasts. Here we report on unique properties of 3D fibroin/gelatin materials, which may rapidly induce expression of adhesion molecules, such as ICAM-1 and VCAM-1, in cultured primary murine embryonic fibroblasts (MEFs). In contrast, two-dimensional (2D) fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions. Interestingly, TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds, while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells. Using selective MAPK inhibitors, we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules. Moreover, we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds, but not on 2D fibroin/gelatin films, suggesting functional reprogramming in stromal cells, when exposed to 3D environment. Finally, we observed significant infiltration of lymphocytes into 3D fibroin/gelatin, but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice. Together our data highlight the important features of fibroin/gelatin scaffolds, when they are produced as 3D sponges rather than 2D films, which should be considered when using these materials for tissue engineering.

Thiophene-2-carboxamide derivatives of anthraquinone: A new potent antitumor chemotype.

The anthraquinone scaffold has long been known as a source of efficacious antitumor drugs. In particular, the various chemical modifications of the side chains in this scaffold have yielded the compounds potent for the wild type tumor cells, their counterparts with molecular determinants of altered drug response, as well as in vivo settings. Further exploring the chemotype of anticancer heteroarene-fused anthraquinones, we herein demonstrate that derivative of anthra[2,3-b]thiophene-2-carboxamide, (compound 8) is highly potent against a panel of human tumor cell lines and their drug resistant variants. Treatment with submicromolar or low micromolar concentrations of 8 for only 30 min was sufficient to trigger lethal damage of K562 chronic myelogenous leukemia cells. Compound 8 (2.5 µM, 3-6 h) induced an apoptotic cell death as determined by concomitant activation of caspases 3 and 9, cleavage of poly(ADP-ribose) polymerase, increase of Annexin V/propidium iodide double stained cells, DNA fragmentation (subG1 fraction) and a decrease of mitochondrial membrane potential. Neither a significant interaction with double stranded DNA nor strong inhibition of the DNA dependent enzyme topoisomerase 1 by 8 were detectable in cell free systems. Laser scanning confocal microscopy revealed that some amount of 8 was detectable in mitochondria as early as 5 min after the addition to the cells; exposure for 1 h caused significant morphological changes and clustering of mitochondria. The bioisosteric analog 2 in which the thiophene ring was replaced with furan was less active although the patterns of cytotoxicity of both derivatives were similar. These results point at the specific role of the sulfur atom in the antitumor properties of carboxamide derivatives of heteroarene-fused anthraquinone.

Effects of Recombinant Spidroin rS1/9 on Brain Neural Progenitors After Photothrombosis-Induced Ischemia.

The existence of niches of stem cells residence in the ventricular-subventricular zone and the subgranular zone in the adult brain is well-known. These zones are the sites of restoration of brain function after injury. Bioengineered scaffolds introduced in the damaged loci were shown to support neurogenesis to the injury area, thus representing a strategy to treat acute neurodegeneration. In this study, we explored the neuroprotective activity of the recombinant analog of Nephila clavipes spidroin 1 rS1/9 after its introduction into the ischemia-damaged brain. We used nestin-green fluorescent protein (GFP) transgenic reporter mouse line, in which neural stem/progenitor cells are easily visualized and quantified by the expression of GFP, to determine the alterations in the dentate gyrus (DG) after focal ischemia in the prefrontal cortex. Changes in the proliferation of neural stem/progenitor cells during the first weeks following photothrombosis-induced brain ischemia and in vitro effects of spidroin rS1/9 in rat primary neuronal cultures were the subject of the study. The introduction of microparticles of the recombinant protein rS1/9 into the area of ischemic damage to the prefrontal cortex leads to a higher proliferation rate and increased survival of progenitor cells in the DG of the hippocampus which functions as a niche of brain stem cells located at a distance from the injury zone. rS1/9 also increased the levels of a mitochondrial probe in DG cells, which may report on either an increased number of mitochondria and/or of the mitochondrial membrane potential in progenitor cells. Apparently, the stimulation of progenitor cells was caused by formed biologically active products stemming from rS1/9 biodegradation which can also have an effect upon the growth of primary cortical neurons, their adhesion, neurite growth, and the formation of a neuronal network. The high biological activity of rS1/9 suggests it as an excellent material for therapeutic usage aimed at enhancing brain plasticity by interacting with stem cell niches. Substances formed from rS1/9 can also be used to enhance primary neuroprotection resulting in reduced cell death in the injury area.

Amides of pyrrole- and thiophene-fused anthraquinone derivatives: A role of the heterocyclic core in antitumor properties.

Heteroarene-fused anthraquinone derivatives represent a class of perspective anticancer drug candidates capable of targeting multiple vital processes including drug resistance. Taking advantage of previously demonstrated potential of amide derivatives of heteroarene-fused anthraquinones, we herein dissected the role of the heterocyclic core in antitumor properties. A new series of naphtho[2,3-f]indole-3- and anthra[2,3-b]thiophene-3-carboxamides was synthesized via coupling the respective acids with cyclic diamines. New compounds demonstrated a submicromolar antiproliferative potency close to doxorubicin (Dox) against five tumor cell lines of various tissue origin. In contrast to Dox, the new compounds were similarly cytotoxic for HCT116 colon carcinoma cells (wild type p53) and their isogenic p53 knockout counterparts. Modification of the heterocyclic core changed the targeting properties: the best-in-series naphtho[2,3-f]indole-3-carboxamide 8 formed more affine complexes with DNA duplex than furan and thiophene analogs, a property that can be translated into a stronger inhibition of topoisomerase 1 mediated DNA unwinding. At tolerable doses the water soluble derivative 8 significantly inhibited tumor growth (up to 79%) and increased the lifespan (153%) of mice bearing P388 lymphoma transplants. Together with better solubility for parenteral administration and well tolerance by animals of the indole derivative 8 indicates prospects for further search of new antitumor drug candidates among the heteroarene-fused anthraquinones.

Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

Neural transplantation is a promising modality for treatment of neurodegenerative diseases, traumatic brain injury and stroke. Biocompatible scaffolds with optimized properties improve the survival of transplanted neural cells and differentiation of progenitor cells into the desired types of neurons. Silk fibroin is a biocompatible material for tissue engineering. Here, we describe thin-film scaffolds based on photocrosslinked methacrylated silk fibroin (FBMA). These scaffolds exhibit an increased mechanical stiffness and improved water stability. Photocrosslinking of fibroin increased its rigidity from 25 to 480 kPa and the contact angle from 59.7 to 70.8, the properties important for differentiation of neural cells. Differentiation of SH-SY5Y neuroblastoma cells on FBMA increased the length of neurites as well as the levels of neural differentiation markers MAP2 and ßIII-tubulin. Growth of SH-SY5Y cells on the unmodified fibroin and FBMA substrates led to a spontaneous phosphorylation of Src and Akt protein kinases critical for neuronal differentiation; this effect was paralleled by neural cell adhesion molecule elevation. Thus, FBMA is an easily manufactured, cytocompatible material with improved and sustainable properties applicable for neural tissue engineering.

New anthra[2,3-b]furancarboxamides: A role of positioning of the carboxamide moiety in antitumor properties.

Derivatives of the anthraquinone (anthracene-9,10-dione) such as doxorubicin, mitoxantrone and others have proved great clinical efficacy for decades. Currently the search in this exceptionally productive chemical class is aimed at optimization of antitumor properties including circumvention of drug resistance. Previously we have reported that heteroarene-fused anthraquinones fused to a 5-membered heterocyclic ring are advantageous in killing drug resistant tumor cells. Herein we present the synthesis and antitumor properties of a series of new anthra[2,3-b]furan-2-carboxamides. Vast majority of new derivatives were similarly cytotoxic to wild type tumor cell lines and their isogenic sublines with P-glycoprotein overexpression and/or p53 inactivation. Comparison of structurally close derivatives varying in their position relative to the furan moiety, that is, furan-3-carboxamide 1vs furan-2-carboxamides 5 and 6, revealed fundamental differences in the cytotoxicity profiles, formation of drug-DNA complexes, efficacy of topoisomerase 1 inhibition and mechanisms of tumor cell death. Together with previous SAR data on the role of individual substituents, these results provide evidence that regioisomerization of anthra[2,3-b]furancarboxamides generates the practically perspective derivatives whose properties may vary significantly.

Radioprotective role of cyanobacterial phycobilisomes.

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called "Great Oxygenation Event" that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis.

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content.

Differences in amino acid sequences of mistletoe lectin I and III B-subunits determining carbohydrate binding specificity.

Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer.

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e6 (the conjugate of chlorin e6 with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e6 against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e6 is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e6. Unlike chlorin e6, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters.

Unsaturated lipids protect the integral membrane peptide gramicidin A from singlet oxygen.

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e6 amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.

Domain structure and conformational changes in rat KV2.1 ion channel.

Voltage-gated potassium Kv2.1 channels are widely distributed in the central nervous system, specifically in neuroendocrine and endocrine cells. Their cytoplasmic C-termini are large and carry out many important functions. Here we provide the first direct structural evidence that each C-terminal part within the Kv2.1 ion channel is formed by two distinct domains (Kv2 and CTA). We expressed and purified two C-terminal truncation mutants of a rat Kv2.1 channel, lacking the entire C-termini or the CTA domain. Single particle electron microscopy was used to obtain three-dimensional reconstructions of purified C-terminal Kv2.1 mutants at 2.0 and 2.4 nm resolution. Comparison of these structures to each other and to the low-resolution EM structure of the full-length Kv2.1 channel revealed the exact locations of cytoplasmic Kv2 and CTA domains within the tetramer. Four Kv2 domains envelop the N-terminal T1 domain. The tetramer of the CTA domains underlies the Kv2-T1 complex and may also affect the channel's surface expression. Subsequent molecular dynamics simulation and homology modeling produced open and closed structural models of the membrane part of the Kv2.1 channel.

Tissue regeneration in vivo within recombinant spidroin 1 scaffolds.

One of the major tasks of tissue engineering is to produce tissue grafts for the replacement or regeneration of damaged tissue, and natural and recombinant silk-based polymer scaffolds are promising candidates for such grafts. Here, we compared two porous scaffolds made from different silk proteins, fibroin of Bombyx mori and a recombinant analog of Nephila clavipes spidroin 1 known as rS1/9, and their biocompatibility and degradation behavior in vitro and in vivo. The vascularization and intergrowth of the connective tissue, which was penetrated with nerve fibers, at 8 weeks after subcutaneous implantation in Balb/c mice was more profound using the rS1/9 scaffolds. Implantation of both scaffolds into bone defects in Wistar rats accelerated repair compared to controls with no implanted scaffold at 4 weeks. Based on the number of macrophages and multinuclear giant cells in the subcutaneous area and the number of osteoclasts in the bone, regeneration was determined to be more effective after the rS1/9 scaffolds were implanted. Microscopic examination of the morphology of the matrices revealed differences in their internal microstructures. In contrast to fibroin-based scaffolds, the walls of the rS1/9 scaffolds were visibly thicker and contained specific micropores. We suggest that the porous inner structure of the rS1/9 scaffolds provided a better micro-environment for the regenerating tissue, which makes the matrices derived from the recombinant rS1/9 protein favorable candidates for future in vivo applications.

Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds.

Non-thermal (low-temperature) physical plasma is under intensive study as an alternative approach to control superficial wound and skin infections when the effectiveness of chemical agents is weak due to natural pathogen or biofilm resistance. The purpose of this study was to test the individual susceptibility of pathogenic bacteria to non-thermal argon plasma and to measure the effectiveness of plasma treatments against bacteria in biofilms and on wound surfaces. Overall, Gram-negative bacteria were more susceptible to plasma treatment than Gram-positive bacteria. For the Gram-negative bacteria Pseudomonas aeruginosa, Burkholderia cenocepacia and Escherichia coli, there were no survivors among the initial 10(5) c.f.u. after a 5 min plasma treatment. The susceptibility of Gram-positive bacteria was species- and strain-specific. Streptococcus pyogenes was the most resistant with 17 % survival of the initial 10(5) c.f.u. after a 5 min plasma treatment. Staphylococcus aureus had a strain-dependent resistance with 0 and 10 % survival from 10(5) c.f.u. of the Sa 78 and ATCC 6538 strains, respectively. Staphylococcus epidermidis and Enterococcus faecium had medium resistance. Non-ionized argon gas was not bactericidal. Biofilms partly protected bacteria, with the efficiency of protection dependent on biofilm thickness. Bacteria in deeper biofilm layers survived better after the plasma treatment. A rat model of a superficial slash wound infected with P. aeruginosa and the plasma-sensitive Staphylococcus aureus strain Sa 78 was used to assess the efficiency of argon plasma treatment. A 10 min treatment significantly reduced bacterial loads on the wound surface. A 5-day course of daily plasma treatments eliminated P. aeruginosa from the plasma-treated animals 2 days earlier than from the control ones. A statistically significant increase in the rate of wound closure was observed in plasma-treated animals after the third day of the course. Wound healing in plasma-treated animals slowed down after the course had been completed. Overall, the results show considerable potential for non-thermal argon plasma in eliminating pathogenic bacteria from biofilms and wound surfaces.

Novel photosensitizers trigger rapid death of malignant human cells and rodent tumor transplants via lipid photodamage and membrane permeabilization.

BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6), that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6) against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6) in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage.

A new gene encoding the ribosome-inactivating protein from mistletoe extracts.

Extracts from mistletoe (Viscum album L.) contain three main toxic proteins--the lectins MLI (also known as viscumin), MLII and MLIII. A catalytic subunit of the mistletoe plant toxic lectin MLIII has been cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII A-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Ribosome-inactivating activity of the recombinant MLIII A-subunit was determined in a cell-free system exhibiting inhibition of endogenous protein synthesis. The comparative analysis of nucleotide and deduced amino acid sequences of the cloned MLIII A and the native MLI A-subunits was performed, revealing the main differences in the primary structure of these proteins. Antigenicity analysis of the MLIII A-subunit has revealed a new epitope D179-E184 that is not present in viscumin. The role of toxic lectins with respect to the immunological properties of mistletoe extracts is discussed.

Importance of Protocol Immunization in Epitope Selectivity of Monoclonal Antibodies Interacting with Binding Subunit of Viscumin.

The application of two immunization protocols and two screening systems has allowed to produce five hybridomas mlb5, mlb6, mlb7, mlb9 and Mbch1, secreting mAbs against different sites of viscumin B-subunit. On the base of mlb9 and Mbch1 hybridomas, the test-system has been developed, able to detect up to 5 ng/ml of viscumin and up to 1 ng/ml of its B-chain. Produced hybridomas and monoclonal antibodies will be used for the studies of intracellular transport of plant toxins. Monoclonal antibody mlb7 will be used for the studies of viscumin interactions with immune system of mammals.