Effect of red seaweed sulfated galactans on initial steps of complement activation in vitro.

The research described here presents data on the effect of galactans of red algae, carrageenans (λ/µ/ν-, κ-, κ/ß-, and ι/κ-types), and agar on complement system activation in normal human serum. The experiments were based on well surfaces coated with triggering agents for binding initiating complement components -C3 and C4. The sulfated galactans inhibited C3 binding to lipopolysaccharide with direct dependence on the sulfation degree of polysaccharides. Sulfation degree was also important in carrageenans' capacity to reduce C4 binding to mannan. However, C4 binding to antibodies was considerably activated by carrageenans, especially with 3,6-anhydrogalactose. The gelling carrageenans were able to block antigen binding centers of total serum IgM and with more intensity than non-gelling. No structural characteristics mattered in ameliorating C5 cleavage by plasmin in extrinsic protease complement activation, but λ/µ/ν- and κ/ß-carrageenans almost completely inhibited C5 cleavage. Thus, galactans participated in cell surface biology by imitating surface glycans in inhibition of C3 binding and mannose binding lectin, but as to the tthe heclassical pathway these substances stimulated complement, probably due to their structure based on carrabiose.

Influence of structural features of carrageenan on the formation of polyelectrolyte complexes with chitosan.

The polyelectrolyte complexes (PEC) of carrageenans (CG)-κ-, κ/ß-, λ-and x-CG with chitosan were obtained. The formation of PEC was detected by Fourier-transform infrared (FTIR) spectroscopy and by centrifugation in a Percoll gradient. The influence of the structural peculiarities of CG on its interaction with chitosan was studied. The results of centrifugation showed that x-CG with a high degree of sulphation (SD) was completely bound to chitosan, unlike low SD κ-CG and κ/ß-CG. Binding constant values showed there was a high affinity of CG for chitosan. CG with flexible macromolecule conformation and high SD exhibited the greatest binding affinity for chitosan. The full-atomic 3D-structures of the PEC κ-CG: chitosan in solution have been obtained by the experiments in silico for the first time. The amino groups of chitosan make the largest contribution to the energy of the complex formation by means of hydrogen and ionic bonds. The most probable complexes have stoichiometries of 1:1 and 1:1.5.

Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes.

It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures. This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S-rolB and 35S-rolC genes in comparison with a non-transformed callus culture. The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells. Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R. cordifolia calluses, whereas ethephon did not. A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only. We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.

[Localization of the antigenic determinants in carcinoembryonic antigen: the role of the carbohydrate component]. / Lokalizatsiia antigennykh determinant v rakovo-émbrional'nom antigene, rol' uglevodnogo komponenta.

The majority of topographic antigenic determinants of the carcinoembryonic antigen (CEA) representing a glycoprotein were shown to contain sugar residues of the CEA carbohydrate chains. Periodate oxidation of CEA followed by reduction afforded a corresponding CEA derivate (CEA-POR), which retained 3% antigenic activity of CEA. In secondary and tertiary structures CEA-POR was proved to be close to CEA pre heated to 80 degrees C. Formation of borate complexes with sugar residues of CEA decreased the CEA antigenic activity to 5% while a but did not affect the spatial structure of its protein core.

[Immunochemical and physico-chemical characteristics of glycoprotein from retroplacental blood having several common antigenic determinants with trophoblastic beta 1-glycoprotein]. / Immunokhimicheskaia i fiziko-khimicheskaia kharakteristika glikoproteina iz retroplatsentarnoi krovi, imeiushchego riad obshchikh antigennykh determinant s trofoblasticheskim beta 1-glikoproteinom.

A glycoprotein (TSG-fs) isolated from retroplacental blood is shown to have common antigenic determinants with trophoblast-specific beta 1-glycoprotein (TSG, SP-1). Antigenic activity of TSG-fs constitute 3.4% of TSG's activity. Like TSG, glycoprotein TSG-fs contains four intramolecular disulfide bonds and belongs to proteins with predominantly beta-structure. CD-spectroscopy data give evidence of high stability of the secondary structure of TSG-fs. Immunochemical identity of TSG-fs with the reduced and carboxymethylated derivative of TSG has been demonstrated. The above data allow to suggest that TSG-fs is a fragment of the TSG molecule.

[Localization of antigenic determinants on a molecule of trophoblast-specific beta 1-glycoprotein]. / Lokalizatsiia antigennykh determinant na molekule trofoblastspetsificheskogo beta 1-glikoproteina.

Spatial localization of antigenic determinants of trophoblast-specific beta I-glycoprotein (TSG) has been elucidated using chemical modifications of the sugar and protein moieties of the molecule. Various deglycosylation procedures of TSG afforded fragments slightly soluble even in the presence of powerful detergents. Treatment of TSG with boric acid and its salts, accompanied with a conformational change of the sugar moiety, failed to alter conformation of the protein portion as evidenced by CD spectral data. This modification was found to increase the antigenic activity of TSG only scarcely. Modification of tryptophane or tyrosine residues of TSG changed spatial structure of the protein portion to can be a considerable loss of the TSG antigenic activity. The data obtained led to the conclusion that antigenic determinants of TSG are localized at the protein portion of the molecule and are topographic. A tryptophane residue is an indispensable constituent of the antigenic determinants.

[The role of tryptophan residues in the antigenic activity of carcinoembryonic antigen]. / Rol' ostatkov triptofana v antigennoi aktivnosti rakovo-émbrional'nogo antigena.

Antigenic determinants of carcino-embryonic antigen (CEA) were spatially located using N-bromosuccinimide modification of tryptophan residues both in native (acetate buffer solution) and unfolded (guanidinium chloride solution) molecule of the antigen. Modification of exposed tryptophan residues failed to alter CEA antigenic activity and conformation of its protein portion as shown by CD spectroscopy. On the contrary, modification of buried tryptophan residues induced conformational changes of CEA protein portion connected with a considerable loss of its antigenic activity. It was shown that CEA antigenic activity depends on spatial structure of its protein moiety.

[Relation between the spatial structure and antigenic activity of trophoblast-specific beta1-glycoprotein]. / Issledovanie vzaimosviazi prostranstvennoi struktury i antigennoi aktivnosti trofoblastspetsificheskogo beta1-glikoproteina.

Temperature- and pH-dependence of spatial structure of a native trophoblast-specific beta-glycoprotein (TSG), its desialated and deglycosylated derivatives, as well as of a fragment obtained by partial acid hydrolysis on the temperature and pH variations has been studied using CD and UV spectroscopy. Within the range 45-50 degrees C a conformational transition of the protein moiety of TSG, leading to partially reversible alterations in tertiary and secondary structures of this glycoprotein after cooling the solution to 20 degrees C has been found out. The results of spectral studies of the spatial structure of the TSG protein component have been compared with the data on antigen activity of native, temperature- and pH-denaturated, desialated, and deglycosilated TSG. It has been concluded that the protein moiety of TSG consists mainly of beta-structures; the greater part of antigen determinants of TSG is topographic and belongs to the protein component of TSG, and only 15% of antigen determinants are not connected with TSG's spatial structure.

[Conformational stability and immunochemical properties of yersinin--a basic protein of the outer membrane of the Pseudotuberculosis microbe]. / Konformatsionnaia stabil'nost' i immunokhimicheskie svoistva iersinina--osnovnogo belka vneshnei membrany psevotuberkuleznogo mikroba.

Using spectroscopic methods (circular dichroism and intrinsic protein fluorescence) and immunoenzyme assay, changes in the spatial and antigenic structure of yersinin, porin from outer membrane of Yersinia pseudotuberculosis, were studied in solutions of ionic and non-ionic detergents at various temperatures and low pH values. Yersinin was shown to retain its secondary structure under various denaturation conditions, the content of regular structural patterns depending on specific action of the denaturation agent. Process of yersinin denaturation similarly to other membrane proteins appears to occur via two structural transitions: dissociation of oligomers and denaturation of monomers. At the first stage changes of quaternary structure accompanied by the loss of so called conformational determinants were observed. Temperature-dependent changes of monomers' tertiary structure affect antigenic activity of yersinin in a smaller degree.

[Interrelation of conformational changes of crustacin and its capacity to bind specifically with carcino-embryonic antigen]. / Vzaimosviaz' konformatsionnykh izmenenii krustatsina i ego sposobnosti spetsificheski sviazyvat'sia s rakovo-émbrional'nym antigenom.

Temperature-, ionic strength-, calcium ion- and pH-dependence of spatial structure of crustacin have been studied using CD and fluorescent spectroscopy. Secondary structure of crustacin was estimated by CD spectra. An irreversible conformational transition of crustacin's protein moiety connected with the loss of CEA-binding activity has been found at ca. 50 degrees C. Crustacin is shown to be calcium-binding protein, stability of the native crustacin conformation being markedly enhanced by calcium ions (1 mM Ca2+ shifted up the transition temperature by approximately 10 degrees C). Calcium binding and ionic strength increase led to alteration of both secondary and tertiary structures of crustacin. The highest CEA-binding activity was observed for the calcium-bond form of crustacin. A lack of specific interaction of crustacin with some saccharides was shown. Interrelation between conformation and immunochemical activity of crustacin is discussed.

[Study of the spatial structure of the protein component of the carcino-embryonic antigen by circular dichroism, Raman spectroscopy and UV-spectroscopy]. / Issledovanie prostranstvennoi struktury belkovogo komponenta rakovo-émbrional'nogo antigena metodami KR, KD i UF-spektroskopii.

The spatial structure features of intact and deglycosylated carcino-embryonic antigen (CEA) have been studied by circular dichroism. Raman and UV-spectroscopy methods in order to elucidate a pattern and localization of CEA immunodominants. The temperature-induced changes in the spatial structure of the protein moiety were compared with data on the CEA immunochemical activity estimated by EIA procedure. A conformational transition was found within the 55-75 degrees range that produced irreversible alterations in the tertiary structure, while the secondary structure could be restored after lowering the temperature to 20 degrees C. Spectral studies of intact and deglycosylated CEA demonstrated that immunochemical activity, at least partly, was associated with the tertiary structure of the CEA protein portion.

[Conformational state of oncoprecipitin cyprein and its immunologic activity]. / Konformatsionnoe sostoianie onkopretsipitina tsipreina i ego immunologicheskaia aktivnost'.

The effect of solution ionic strength, calcium ion concentration, and temperature on spatial structure of cyprein was examined by CD, UV, and fluorescence spectroscopy. The secondary structure of the cyprein molecule was calculated from CD spectra, and the prevalence of the beta-structure (85%) was shown. An irreversible conformational transition in the range 55-60 degrees C was found, which reduces the binding activity of cyprein in interaction with carcinoembryonic antigen (CEA) and anti-cyprein antibodies. In the latter case, the binding activity was reversibly restored. Cyprein was shown to be a calcium-binding protein. Binding of calcium by cyprein and increasing the ionic strength of solution affect only tertiary structure of the protein. At an ionic strength of solution close to physiological conditions, calcium-bound cyprein shows maximum binding to CEA and anti-cyprein antibodies. It was shown by difference UV spectroscopy that cyprein does not interact specifically with the monosaccharides of the carbohydrate chains of CEA: fucose, mannose, galactose, and N-acetylglucosamine.

[Effect of a lipopolysaccharide on the conformational state and functional activity of a Yersinia pseudotuberculosis porin]. / Vliianie lipopolisakharida na konformatsionnoe sostoianie i funktsional'nuiu aktivnost' porina iz Yersinia pseudotuberculosis.

Changes in the structure and functional activity of porin, a protein from Yersinia pseudotuberculosis, resulting from the removal of lipopolysaccharide (LPS) normally bound with the protein were studied. The treatment of LPS-containing porin with a 30% SDS solution led to an LPS-free protein that, according to the SDS-PAGE, remained to be a trimer. It was shown by CD and UV spectroscopies and intrinsic protein fluorescence that the removal of LPS caused only conformational changes in the porin secondary and tertiary structures. The LPS-free porin folded into a completely beta-structured protein aggregate. The bilayer lipid membrane technique showed that the pore-forming activity of the LPS-free porin decreased, and its concentration should be increased by two orders of magnitude to achieve the same effect. Incubation of the LPS-free porin with LPS led to a porin-LPS complex and affected the character of the protein functional activity. The treatment of the LPS-free porin by octyl glucoside, a nonionic detergent, resulted in the restoration of the protein pore-forming activity. It was suggested that the LPS and detergent provide a definite protein conformation necessary for its functioning.

[Photoinduced enzymatic epimerization of glucose]. / Fotoindutsirovanaia fermentativnaia épimerizatsiia gliukozy.

The possibility was examined whether weak external electromagnetic radiation can affect the parameters of enzymic reactions. It was found that, in a system involving an aqueous solution of concanavalin A, glucose, and erbium salt exposed to helium-neon laser light of a wavelength of 633 nm, epimers of glucose: allosa, mannose, and galactose are formed. The effect observed was found to represent a photoinduced enzymic reaction in which the protein dissolved in water converts the glucose associated with it into epimers by the action of electromagnetic radiation. The photoepimerase activity of concanavalin A was established for the first time.

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus.

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked ß-D-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-D-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.

Comparative study of carrageenans from reproductive and sterile forms of Tichocarpus crinitus (Gmel.) Rupr (Rhodophyta, Tichocarpaceae).

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.

Trophoblast-specific beta 1-glycoprotein: its spatial structure and localization of antigenic determinants.

Effects of temperature and pH on the spatial structures of trophoblast-specific beta 1-glycoprotein (TSG) and its various derivatives and fragments have been studied by circular dichroic spectroscopy. The spatial organization of the protein portion of TSG derivatives, as revealed by the spectroscopic evidence, has been discussed with respect to the antigenic activity of the species studied. We concluded that the TSG protein portion consists mainly of a beta-structural type. The antigenic determinants were shown to be topographic, and are preferentially localized in the protein portion; only about 15% of the TSG antigenic activity failed to bear a direct relation to the spatial structure. The antigenic determinants seemed to include a tryptophan residue.

Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.

Interaction of porin from Yersinia pseudotuberculosis with lipopolysaccharides. Effect of ionic strength, pH, and divalent cations on the binding parameters.

Interaction of the pore-forming protein (porin) from Yersinia pseudotuberculosis with S- and R-forms of the endogenous lipopolysaccharide (LPS) was studied at various ionic strengths (20-600 mM NaCl), concentrations of divalent cations (5-100 mM CaCl2, MgCl2), and pH values from 3.0 to 9.0. The interaction of the R-LPS with porin has been shown in all experimental conditions to be in consensus with the model suggesting binding at independent sites of two types. S-LPS binds to interacting sites of relatively high affinity and to independent sites of low affinity at all pH values examined and at low NaCl concentration. The cooperative interaction of the S-LPS and porin is not observed at high ionic strength and in divalent cation-free medium. The number of binding sites of porin and association constants (Ka) for both LPS forms decrease significantly on increasing the solution ionic strength. The Ka values for the R- and S-LPS change oppositely on changing the pH: the Ka value for the R-LPS is maximal (Ka = 6.7 x 10(5) M-1), but that for S-LPS is minimal (Ka = 0.4 x 10(5) M(-1) at pH 5.0-5.5. The number of high-affinity and low-affinity binding sites for both LPS forms is maximal at pH 5.0-5.5. In this case, the numbers of high- and low-affinity sites for R-LPS are 3 and 10, respectively, and those for the S-LPS are 7 and 20, respectively. These data suggest an important role of electrostatic interactions on binding of LPS to porin. The contribution of conformational changes of the ligand and protein and hydrophobic interactions are discussed.

Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway.

It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca2+ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca2+ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca2+-dependent NADPH oxidase pathway.