Obtaining biologically active substances from Amaranthus caudatus L. seeds in one technological cycle.

Nowadays, amaranth is a valuable multipurpose crop and a source of a number of very important biologically active substances. The aim of this study was to develop a comprehensive scheme for obtaining fatty oil, triterpenoids and lectin from the seeds of Amaranthus caudatus L. in one technological cycle. Two variants of the lectin and triterpene compound purification method from amaranth seeds were tested. It was determined that the extraction of triterpene compounds should be carried out after purification of the lectin from degreased seeds. The rationality of this sequence of technological operations is explained by the lability of the lectin and the insolubility in water of triterpene compounds from amaranth seeds. The study also presents a scheme for obtaining squalene from amaranth oil by chromatography on silica gel and proposes a more effective affinity sorbent for purification of the lectin. The use of such a sorbent also opens up the possibility of preserving other water-soluble substances from amaranth seeds. The physicochemical characteristics and carbohydrate specificity of the lectin are described and new data on the results of the interaction of lectin with human and animal erythrocytes are given. The obtained results are discussed in the light of the complex use of raw materials.

Isolation and identification in human blood serum of the proteins possessing the ability to bind with 48 kDa form of unconventional myosin 1c and their possible diagnostic and prognostic value.

We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Use of specific polysaccharide-immobilized monodisperse poly(glycidyl methacrylate) core-silica shell microspheres for affinity purification of lectins.

Immobilization of polysaccharides (yeast mannan and gum arabic) on the macroporous poly(glycidyl methacrylate) monodisperse microspheres coated with silica (SiO2 )-containing amino groups on the surface was used to prepare affinity sorbents for lectin purification. The efficiency of isolating mannose specific Pisum sativum lectin was demonstrated on sorbent with immobilized yeast mannan and that of galactose specific Glycine hispida lectin on sorbent with immobilized gum arabic. The microspheres with immobilized polysaccharides can be used for selecting an affinity sorbent for purification of other mannose- and galactose-specific lectins. In contrast to yeast mannan, the gum arabic immobilized on the microspheres possesses much narrower specificity and is suitable for purification of only those galactose specific lectins which interact well with l-rhamnose or l-arabinose. The synthesized macroporous particles are capable of immobilizing 50 mg of polysaccharide per 1 g of the matrix, which is 10 times higher than the capacity of epoxy-activated Sepharose 6B. That makes it possible to obtain the same lectin quantity using a column of 10 times smaller volume. Another advantage of novel affinity sorbents comparing corresponding Sepharose gels is the possibility of sorbent drying after use.

Two-step chromatography purification of IgGs possessing sialidase activity from human blood serum.

Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G-Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin-Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum.

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients.

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

Lectin purification from carp roe (Cyprinus carpio L.), investigation of its carbohydrate specificity and application in histochemistry.

A method of lectin purification from carp roe (CCRA) was elaborated, which includes affinity chromatography on cross-linked ovomucoid and copolymers of polyvinyl alcohol and blood group-specific substances. That allowed obtaining lectin with electrophoretic purity and yielding of ˜ 42 mg÷kg roe. Electrophoresis in 15% polyacrylamide gel in the presence of ß-mercaptoethanol showed one band with molecular mass ˜ 15 kDa, whereas in the absence of ß-mercaptoethanol, CCRA exposed band with molecular mass ˜ 60 kDa. The resulting lectin was thermostable, withstanding heating to 75°C for 15 minutes, without noticeable loss of hemagglutinating activity. Gel column chromatography on Toyopearl HW-55 determined the lectin molecular weight of 120±3 kDa. For the lectin activity, divalent metal ions (Ca2+ and Mg2+) were not necessary. CCRA showed the best agglutination titer with pigeon erythrocytes, weaker - with rabbit and dog erythrocytes, and significantly weaker - with human and rat erythrocytes. CCRA lectin was specific to N-acetyl-D-galactosamine and D-galactose group carbohydrates. The best lectin activity inhibition possessed alkaline phosphatase of calf intestine and fetuin. CCRA exposed highest affinity to complex oligosaccharide similar to the receptor of Phaseolus vulgaris erythroagglutinin (PHA-E). A comparative study on the histochemical specificity of CCRA and PHA-E using specimens of normal tissues, and that of colon neoplasia, showed similar, yet not identical binding properties. CCRA lectin rather differentially labeled adenoma and adenocarcinoma of colon, which suggests its prospective applicability in diagnostic histopathology.

Lectin purification from fruiting bodies of brown roll-rim fungus, Paxillus involutus (Fr.) Fr., and its application in histochemistry.

A lectin (agglutinin) from fresh fruit bodies of the brown roll-rim fungus [Paxillus involutus (Fr.) Fr.] has been purified with output approx. 60 mg÷kg of raw material. Method of purification included the sedimentation of viscous polysaccharide by ethanol, removal of ethanol by dialysis, ion-exchange chromatography on DEAE-Toyopearl and affinity chromatography on Sepharose 6B column with immobilized mannose-specific Polygonatum multiflorum lectin. The obtained lectin preparation (abbreviated PIFA) is a glycoprotein with 6.5±1% carbohydrates, molecular mass of 64 kDa, consisting of four identical subunits. Lectin interacted only with N-acetyl-lactosamine and glycoproteins that contained Galß1-4GlcNAc disaccharide moieties; agglutinated erythrocytes of dog, sheep and horse, but not of humans. The specificity of PIFA binding to tissue samples of the rat has been investigated. Lectin selectively reacted with gastric parietal cells, submandibular salivary gland duct cells. In the kidney, PIFA labeled epithelial cells of renal tubules, collecting ducts, nuclei of podocytes and mesangiocytes. It was also revealed selective lectin binding to Purkinje cells of cerebellum. Brush border of absorptive cells in small intestine was also strongly reactive, while goblet cells both in small and large intestine were completely negative. Considering similarities in carbohydrate specificity of Paxillus involutus (PIFA) and Ricinus communis agglutinin (RCA-120), histochemical reactivity of these two lectins was compared. It was similar, yet not identical: differences included absence of PIFA binding to the brush border of renal tubules, higher interaction with absorptive cells of the small intestine, lower background staining of cerebellar cortex and renal corpuscles. A conclusion was made that due to the unique carbohydrate specificity PIFA lectin can cover prospective position in experimental histochemistry and diagnostic histopathology comparable to PNA (Peanut agglutinin) and SNA (Sambucus nigra agglutinin).

Diabetic alteration versus postnatal maturation of rat kidney glycoconjugates--comparative detection by lectin probes.

A panel of ten lectins with different carbohydrate specificities, including three original lectin preparations (MPFA, LABA, and LVA), was used for the investigation of rat kidney glycoconjugate remodeling during postnatal morphogenesis, and the findings were compared with the impairments seen in streptozotocin-induced diabetic nephropathy. Postnatal morphogenesis was accompanied by the accumulation and generalization of DMan, LFuc, and NeuNAc, with simultaneous reduction of DGal and DGalNAc sugar determinants and enhanced heterogeneity of renal microstructure. The most significant redistribution of lectin receptor sites was detected between postnatal days 1 and 20. Beginning from postnatal day 20, renal corpuscles showed selective MPFA, WGA, and RCA labeling. Stabilization of carbohydrate determinants on postnatal day 60 coincided with rat kidney maturity. Diabetic nephropathy induced carbohydrate remodeling reciprocal to that seen during postnatal development, that is, enhanced exposure of DGal and DGalNAc, reduced reactivity of DMan, LFuc, and NeuNAc determinants, and increased lectin labeling of renal tubule brush borders. These results extend the existing data on rearrangement of rat kidney glycoconjugates under physiological and pathological conditions, as well as demonstrate the applicability of three original lectin preparations in glycoconjugate histochemistry.

A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase.

A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21-32]. The physico-chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin-induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l-amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l-amino acid oxidase.