Polyelectrolyte complex formation using alginate and chitosan

Polyelectrolyte complexes (PECs) of alginate and chitosan were formed by addition of 0.1% alginate solution (pH 6.5) to 0.1% chitosan solution (pH 4.0), and by adding the chitosan solution to the alginate solution under high shearing conditions. Variations in the properties of the polymers and the preparation procedure were studied, and the resultant PEC size, zeta potential (Zp), and pH were determined using dynamic light scattering (DLS), electrophoresis and by measuring turbidity and pH. Tapping mode atomic force microscopy (AFM) was used to examine some of the complexes. The particle size was decreased as the speed and diameter of the dispersing element of the homogenizer was increased. The net charge ratio between chitosan and alginate, and the molecular weights (M_W) of both the alginate and chitosan samples were the most significant parameters that influenced the particle size, Zp, and pH. The mixing order also influenced the size of the PECs, however, the Zp and pH were not affected by the mixing order. The stability of the complexes was investigated by incubation at an elevated temperature (37 °C), storage for one month at 4 °C, alteration of the pH of the PEC mixture, and addition of salt to physiological ionic strength (0.15 M NaCl). The properties of the PEC could be affected according to the molecular properties of the polyelectrolytes selected and the preparation procedures used. The resultant PEC sizes and properties of the complex were rationalised using a core-shell model for the structure of the complexes.     

Cationic oligopeptides modified with lipophilic fragments: Use for DNA delivery to cells

Cationic oligopeptides, including the amphipathic -helical peptides, are applied to the targeted delivery of DNA to eukaryotic cells due to their DNA-compacting properties and the ability to destabilize the cell lipid bilayer in some cases. We synthesized the peptides differing in the number and location of residues of decanoic acid covalently attached to Lys residues in order to combine the DNA-binding and the membrane activities in a single molecule. We chose peptide structures that assisted in the formation of helices. The DNA-binding ability of the peptides and the membrane activity of their complexes with DNA were shown to depend on the structure. The study of erythrocyte hemolysis by complexes with DNA of the pCMV LacZ plasmid and the peculiarities of transfection of these complexes revealed a correlation between the hemolytic activity and the expression level of the lacZ gene in cells.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 22–30.Original Russian Text Copyright © 2005 by Guryanov, Vlasov, Lesina, Kiselev, Baranov, Avdeeva, Vorobev.     

Some X-Ray Diffraction Parameters of Liquid-Crystalline Dispersions of Nucleic Acid–Chitosan Complexes

Liquid-crystalline dispersions of nucleic acid–chitosan complexes (NA–chitosan) possess optical and X-ray diffraction properties different from those of classical cholesterics. It is possible that positive charge distribution (distance between charges, chitosan conformation, etc.) in the chitosan polymeric chain interacting with NA is a factor controlling the spatial structure of the resulting dispersions.     

Formylation of porphyrin platinum complexes

The formylation reaction of platinum complexes of -unsubstituted porphyrins was studied. The interaction of deuteroporphyrin IX derivatives with the Vilsmeyer reagent led to the selective formylation of their macrocycles in the position. The resulting formyl derivatives of the porphyrins are of interest for fluorescent immunoassay.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 103–107.Original Russian Text Copyright © 2005 by Rumyantseva, Konovalenko, Nagaeva, Mironov.     

In vitro antifungal activity of metal complexes of a quaternized chitosan derivative with copper ions

Antifungal activity of synthetic metal complexes of quaternized N-(propyl) chitosan derivatives with Сu(II) against yeastlike (Saccharomyces cereviseae, Rodothorula rubra, and Candida albicans) and mycelial fungi (Fusarium oxysporum, Alternaria alternata, Cladosporium herbarum) was studied. In vitro application (at 250?500 μg/mL) of the metal complex of quaternized N-(propyl) derivative of low-molecular chitosan with 53% substitution and 1.3% copper ions proved efficient against F. оxysporum, one of ten most common fungal plant pathogens. Water-soluble quaternized N-(propyl) chitosan derivatives with 40?58% degree of substitution were synthesized using glycidyltrimethylammonium chloride under optimally adjusted conditions. Metal complexes of the chitosan derivative with 53% degree of substitution with Сu(II) ions were obtained by dialysis. The quantity of copper ions in the metal complexes was determined by atomic emission spectrometry. The structure of chitosan derivatives was confirmed by spectral analysis (IR, ¹H NMR).     

Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 μg/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-l-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers l-lysine and d-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺. Na polygalacturonate and Na poly-l-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentration-dependent precipitation of chitosan by Na poly-l-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca²⁺ and Na polygalacturonate.     

Synthesis and antimicrobial activity of the Schiff base from chitosan and citral

Chitosan, a biocompatible, biodegradable, non-toxic polymer, is prepared from chitin, which is the second most naturally occurring biopolymer after cellulose. The Schiff base of chitosan was synthesized by the reaction of chitosan with citral working under high-intensity ultrasound. The effect of the molar ratio of chitosan to citral, reaction time, and temperature on the yield has been investigated. The optimal conditions were a temperature of 50 °C, a molar ratio of chitosan to citral of 1:6, and a reaction time of 10 h. The maximum yield achieved was 86.4% under optimum conditions. The structure of the Schiff base was characterized by FTIR spectroscopy, elemental analysis, and X-ray diffraction studies. The strong peaks at 1648.3 and 1610.6 cm⁻¹ are due to CN and CC stretching vibrations. The results confirmed that amino groups on chitosan reacted with citral to form the Schiff base. The antimicrobial activities of chitosan and Schiff base of chitosan were investigated against Escherichia coil, Staphylococcus aureus, and Aspergillus niger. The results indicate that the antimicrobial activity of the Schiff base increases with an increase in the concentration. It was also found that the antimicrobial activity of the Schiff base was stronger than that of chitosan.     

Chitosan isolation from the chitosan-glucan complex of fungal cell wall using amylolytic enzymes

The chitosan/glucan complex isolated from the mycelia of the fungus, Gongronella butleri USDB 0201 can be cleaved with a heat-stable -amylase at 65 °C for 3 h. This results in the removal of the glucan side chain and gives a chitosan solution with 100 times lower turbidity. It is proposed that chitosan and glucan chains are bound by an (1 to 4) glucosidic bond. Both fungal chitosan and fungal glucan have been purified separately.     

Effect of Intercalators on the Properties of Liquid-Crystalline Dispersions of Nucleic Acid–Chitosan Complex

Molecules of deoxyribonucleic acid and synthetic polydeoxyribonucleotides (NA) in the particles of liquid-crystalline dispersions resulting from interaction with chitosan are accessible to interaction with intercalators. The intercalation is accompanied by alteration in the direction of spatial twist of cholesterics of NA–chitosan complexes. This effect is absent in the case of classical cholesterics produced from NA molecules via phase exclusion, i.e., the cholesteric structure of NA–chitosan complex is very labile as distinct from classical cholesteric NA.     

Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction

An improved method is described for the production of chitosan from mycelia of the fungus Gongronella butleri, grown by solid substrate fermentation on sweet potato. The chitosan was extracted subsequently by 11 M NaOH at 45 °C, and 0.35 M acetic acid at 95 °C. The resulting extract was clarified using a heat-stable, commercial -amylase. The yield (4–6 g/100 g mycelia) and relative number average molecular weight (44–54 kDa) of the chitosan increased with increasing duration of fungal growth up to the sixth day.     

Chitosan-Mediated siRNA Delivery <Emphasis Type="Italic">In Vitro</Emphasis>: Effect of Polymer Molecular Weight,Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.     

Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions

Chitosan particles were functionalized with ferulic acid (FA) and ethyl ferulate (EF) as substrates using laccase from Myceliophtora thermophyla as biocatalyst. The reactions were performed with chitosan particles under an eco-friendly procedure, in a heterogeneous system at 30 °C, in phosphate buffer (50 mM, pH 7.5).The FA-chitosan derivative presented an intense yellow-orange color stable while the EF-chitosan derivative was colorless. The spectroscopic analyses indicated that the reaction products bound covalently to the free amino groups of chitosan exhibiting a novel absorbance band in the UV/Vis spectra between 300 and 350 nm, at C-2 region by the duplication of C-2 signal in the ¹³C NMR spectrum, via Schiff base bond (NC) exhibiting novel bands in the FT-IR spectrum at 1640 and 1620 cm⁻¹. Additionally, antioxidant capacities of chitosan derivatives showed that the chitosan derivatives presented improved antioxidant properties, especially for FA-chitosan derivative (EC₅₀ were 0.52 ± 0.04, 0.20 ± 0.02 mg/ml for DPPH and ABTS⁺ scavenging, respectively).     

Synthesis of diamine maleyl chitosans, and in vitro transfection studies

Three novel diamine-modified chitosan derivatives were synthesized from N-maleyl chitosan via Michael addition reaction with 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane, respectively. These chitosan derivatives exhibited well binding ability of condensing plasmid DNA to form complexes with size ranging from 150 to 500 nm when the chitosan derivative/DNA weight ratios were above 10. The complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The cytotoxicity of the chitosan derivatives presented a dependence on their side-chain structures. The gene transfection experiments were evaluated in 293 T and HeLa cells. The data obtained demonstrated that the gene transfection efficiencies of these chitosan derivatives were better than that of chitosan, suggesting these chitosan derivatives as potential gene vectors in vitro.     

Antenna organisation in the purple bacterium Rhodopseudomonas acidophila studied by fluorescence induction

The photosynthetic membrane of the purple bacterium Rhodopseudomonas (Rps.) acidophila is composed of reaction centers (RCs) which are surrounded by closely connected light harvesting complexes (LH1) and peripheral light-harvesting complexes (LH2). Both LH1 and LH2 – which bind the antenna pigments between -, -heterodimers – form rings composed of an integer number of -, -subunits. Here we use the sigmoidicity of fluorescence induction curves to probe the excitonic connectivity of RCs in order to gain information on the structural arrangement of these LH complexes in the natural chromatophore membrane. The data exclude models of the Rps. acidophila photosynthetic unit that assume aggregates of RC-LH1 complexes or linear chains of RC-LH1 complexes to which LH2 complexes are attached on the periphery. Rather, they support the model suggested by Papiz et al. ((1996) Trends in Plant Science 1: 198–206) in which peripheral light-harvesting rings tightly surround each core complex (LH1-ring with the RC inside) circumferentially.     

Chitosan membranes modified by contact with poly(acrylic acid)

In this work chitosan membranes modified by contact with poly(acrylic acid) (PAA) aqueous solution at two different temperatures (25 °C and 60 °C) were obtained. The pure chitosan (CS) membranes, as well as those treated with PAA (CSPAA_25 and CSPAA_60) were characterized by FTIR-ATR, water sorption capacity, thermal analysis (TG/DTG), and scanning electron microscopy (SEM). In addition, in vitro permeation experiments were carried out using metronidazol and sodium sulfamerazine aqueous solutions at 0.1% and 0.2% as model drugs. FTIR-ATR results showed the presence of absorption bands of and COO⁻ indicating the formation of a polyelectrolyte complex between chitosan and poly(acrylic acid). The results also indicated that PAA penetrates deeper into the membrane at higher temperature (60 °C), forming a thicker complex layer. Polyelectrolyte complex formation as well as the influence of treatment temperature was confirmed by lower hydrophilicity, higher thermal stability, and lower permeability of the treated membranes. The results show that the methodology used is a simple and very efficient way to drastically change some membrane properties, especially their permeability.     

Studies on structure activity relationship of some dihydroxy-4-methylcoumarin antioxidants based on their interaction with Fe(III) and ADP

Three dihydroxy-4-methylcoumarin (DHMC) derivatives, namely 7,8-DHMC, 6,7-DHMC and 5,7-DHMC alone and complexed with Fe (III) and ADP have been tested for their antioxidative potential. Chemical speciation studies and formation constants reveal the formation of strong DHMC–Fe–ADP (1:1:1) ternary complex. In vitro studies were done for their antioxidative property by scavenging the superoxide radicals (O ₂ ^– ) generated by xanthine + xanthine oxidase (XO) reaction. The IC₅₀ values for 7,8-DHMC, 6,7-DHMC and 5,7-DHMC and their ternary complexes with Fe (III)–ADP worked out to be 34.0 M, 62.0 M, 8.80 mM and 10.5, 11.5 and 148.5 M, respectively. The results indicate that O ₂ ^– scavenging potential of all the three DHMCs increased significantly after forming the ternary complex with Fe(III) and ADP. The structure activity relationship studies suggest that the introduction of hydroxyl group at 7th and 8th positions in the coumarins, irrespective of Fe(III)–ADP complexation, increases the antioxidative efficacy. No change in uric acid production in the reactions done for all studies further reveals that the coumarin derivatives and their complexes were the only causative factors for O ₂ ^– scavenging and not the suppression of the enzyme, xanthine oxidase.Published online: March 2005     

Effects of root temperature on uptake of nitrate and ammonium ions by barley grown in flowing-solution culture

Summary Barley plants (Hordeum vulgare L.) grown from seed for 28 days in flowing solution culture were subjected to different root temperatures (3, 5, 7, 9, 11, 13, 17, 25°C) for 14 days with a common air temperature of 25/15°C (day/night). Uptake of NH₄ and NO₃ ions was monitored separately and continuously from solutions maintained at 10 M NH₄NO₃ and pH 6.0. Effects of root temperature on unit absorption rate , flux and inflow were compared. After 5 days , and increased with temperature over the range 3–11°C for NH₄ ions and over the range 3–13°C for NO₃ ions, with little change for either ion above these temperatures. Q₁₀ temperature coefficients for NH₄ ions (3–13°C) were 1.9, 1.7 and 1.6 for , and respectively, the corresponding values for NO₃ ions being 5.0, 4.5 and 4.6. For both ions, , and changed with time as did their temperature dependence over the range 3–25°C, suggesting that rates of ontogenetic development and the extent of adaptation to temperature may have varied among treatments.     

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2±0.2 M and 11.2±0.2 M at 5 °C and 20°C respectively.This revised version was published online in October 2005 with corrections to the Cover Date.     

Seven new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from colubrid snakes of Guatemala and a discussion of what to call ellipsoid tetrasporocystic, dizoic coccidia of reptiles

During a survey of Guatemalan herpetofauna in the summers of 1998–2000, 29 presumed new species of Eimeria Schneider, 1875 were found, seven of which have a distinct elongate-ellipsoidal shape (L/W ratio ≥ 1.7) and are described herein. Six of the seven new species are similar in oöcyst length, width and L/W ratio and sporocyst length, width and L/W ratio, lack a micropyle, oöcyst residuum, Stieda body, sub- and parastieda bodies, have a polar granule and sporocyst residuum, and their sporocysts appear to have dehiscence sutures. The seventh is slightly smaller and has sporocysts with a Stieda body. The new species are: E. coniophanes n. sp – whose sporulated oöcysts from Coniophanes fissidens are 29.2×14.9 (27–31×13–16) m, with sporocysts m; E. coniophis n. sp. –from Conophis lineatus are 32.0×16.5 (30–34×14–18) m, with sporocysts m; E. dryomarchoni n. sp. – from Drymarchon corais are 32.2×17.7 (31–34×17–19) m, with sporocysts m; E. leptophis n. sp. – from Leptophis mexicanus are 29.5×17.0 (28–31×16–18) m, with sporocysts m; E. oxybelis n. sp. – from Oxybelis aeneus are 31.8×16.5 (29–33×15–18) m, with sporocysts m; and E. scaphiodontophis n. sp. – from Scaphiodontophis annulatus are 30.0×15.3 (28–33×14–16) m, with sporocysts m. Sporulated oöcysts of E. siboni n. sp. from Sibon nebulata are 24.3×14.2 (21–27×13–16) m, with sporocysts m and with a Stieda body. We conclude that until all aspects of each life-cycle are known, it is prudent at this time to name all tetrasporocystic dizoic coccidia from snakes as members of Eimeria rather than place some of them in Choleoeimeria Paperna & Landsberg, 1989.