Carrageenan gel beads for echinochrome inclusion: Influence of structural features of carrageenan.

Carrageenan (CRG) and carrageenan/chitosan (CH) gel beads (CRG/CH) were prepared as a release delivery system for echinochrome A (Ech). According to spectral data, the Ech was dispersed in the polymer matrix, interacted with CRG, was not oxidised, and remained stable after encapsulation in CRG beads. Carrageenan beads containing Ech were coated with CH by layering. The influence of the structural features of CRG on the formation of beads and the beads morphology, swelling behaviour, mucoadhesive properties and drug release were evaluated. The polysaccharide matrices with Ech showed different swelling characteristics depending on the pH of the medium and the structure of the CRG used. The slow drug release from polysaccharide matrixes was observed for κ- and κ/ß-CRG beads, that contained 3,6-anhydro-α-d-galactopyranose units and had high molecular weight. The obtained results showed the prospects of using polysaccharide beads to include Ech.

OmpF porin from Yersinia ruckeri as pathogenic factor: Surface antigenic sites and biological properties.

Bacterium Yersinia ruckeri as a pathogen induces causative agent of intestinal fish disease called enteric redmouth disease (ERM) is known. In this study, outer membrane OmpF porin from the Y. ruckeri (YrOmpF) has been identified as a pathogenic factor which affects host macrophage activation and life cycle of eukaryotic cells. Using synthetic peptides corresponding to the sequences of the outer loops of YrOmpF L1 loop of the porin is most involved in the structure of B epitopes on the surface of the microbial cell it was found. T epitopes of the isolated YrOmpF trimer not only by linear, but also by discontinuous determinants, which is due to the secondary structure of the protein are represented. It was shown that YrOmpF was twice more cytotoxic to THP-1 cells (human monocytes, cancer cells) in comparison with CHH-1 cells (Oncorhynchus keta cardiac muscle cell, non-cancer cells). It was found YrOmpF induce cell cycle S-phase arrest in both normal CHH-1 and cancer THP-1 cells. In the cancer cells observed effect was most pronounce. In addition, we have observed an induction of apoptosis in THP-1 cell line treated with YrOmpF for 48 h at IC50 (48.6 µg/ml). Significant cytotoxic effect of YrOmpF on primary mouse peritoneal macrophages been detected as well. Of note, co-incubation of macrophages with anti-YrOmpF antibodies could decrease the amount of lactate dehydrogenase, while the number of living cells significantly increased. YrOmpF stimulates the activity of the phagocytic bactericidal systems especially of the oxygen-independent subsystem it was found. Antibodies against YrOmpF decreased MPO release and CP synthesis by peritoneal macrophages and increased their viability.

Mucoadhesive properties of sulphated polysaccharides carrageenans from red seaweed families Gigartinaceae and Tichocarpaceae.

The mucoadhesive properties of different types of carrageenan (kappa-, kappa/beta-, iota/kappa- and lambda-CRGs) isolated from red seaweed families Gigartinaceae and Tichocarpaceae collected on the Pacific coast were studied. We examined the interaction between CRGs and pig stomach mucin in dilute aqueous solutions using a set of methods. Measurements of the dynamic light scattering of mucin in the presence of CRG showed that the polysaccharides cause aggregation of mucin particles, as confirmed by microscopy data. The addition of CRGs to solutions of mucin resulted in the formation of a mixture that changed the charge of mucin, especially in the case of kappa- and kappa/beta-CRGs. The interaction between CRG and porcine gastric mucin in the presence of various additives confirmed that hydrogen bonds and electrostatic interactions are complemented when CRG and mucin are mixed in an aqueous medium, which is also confirmed by in vitro methods based on measurements of work of adhesion and shear stress. Kappa- and kappa/beta-CRGs that contain 3,6-anhydro-α-d-galactopyranose chains (DA) have high molecular weight and exhibit a high density of available hydrogen bonding groups able to interact more strongly with mucin glycoproteins.

Peculiarities of thermal denaturation of OmpF porin from Yersinia ruckeri.

Irreversible denaturation of membrane proteins in detergent solutions is similar to unfolding of water-soluble multidomain proteins and represents a complex, multistage process. Pore-forming proteins of Gram-negative bacteria are heat-modifiable proteins, i.e., proteins altering their molecular forms (trimers or monomers), and accordingly, their electrophoretic mobilities depending upon denaturation conditions. There are still some contradictory data on the peculiarities of the conformational changes in the porin structure with temperature. Some authors demonstrated the loss of the porin trimeric structure only after unfolding of monomer subunits. Other researchers initially observed the dissociation of porin oligomers into the folded monomers. Using SDS-PAGE, spectroscopic methods and differential scanning calorimetry, a detailed study of thermally induced changes in the spatial structure of OmpF porin from the fish pathogen Yersinia ruckeri (Yr-OmpF) was carried out. The data obtained allowed us to conclude unambiguously that changes in the spatial structure of the monomers of Yr-OmpF precede the dissociation of the porin trimer.

pH-Dependent properties of ion channels formed by N-terminally glutamate substituted gramicidin A in planar lipid bilayers.

The N-terminally glutamate substituted analogue of the pentadecapeptide gramicidin A [Glu1]gA has been previously described as a low-toxic uncoupler of mitochondrial oxidative phosphorylation and neuroprotector. Here, we studied ion channel-forming activity of this peptide in planar bilayer lipid membranes (BLMs). [Glu1]gA exhibited an ability to induce both macroscopic current and single channels in a broad pH range, albeit with a lower potency than the parent gramicidin A (gA). Single-channel recordings in 1M KCl at pH about 4 showed channel openings of one type with the conductance (about 26pS), similar to that of gA, and the lifetime (40ms), much shorter than that of gA. By contrast, two populations of channels were found at pH9, one of which had much longer duration (several seconds) and lower conductance (3.5-10pS). Autocorrelation function of the current noise of [Glu1]gA revealed a marked shift towards longer correlation times upon alkalinization. The sensitized photoinactivation technique also revealed substantial differences in [Glu1]gA conducting properties at alkaline and acidic pH, in particular deceleration of the photoinactivation kinetics and a sharp decrease in its amplitude upon alkalinization. A double-logarithmic plot of the concentration dependence of [Glu1]gA-induced BLM conductance had the slope of about 3, which pointed to peptide aggregation in the membrane. The data were discussed in relation to pH-dependent aggregation of [Glu1]gA, resulting from deprotonation of the glutamate side chain at alkaline pH.