Influence of chitosan derivatives on the development of phage infection in theBacillus thuringiensis culture

The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infectionof Bacillus thuringiensis by phage 1–97 A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 μg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate andN-succinate-6-O-sulfate did not inactivate the phage, did not influence bacterial growth, and did not inhibit the process of viral infection.     

Effect of chitosan derivatives on the reproduction of Coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosan N-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 in Escherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited bactericidal activity. The efficiency of inhibition of viral infection and the bactericidal activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or bactericidal activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability of E. coli may also be due to the bactericidal effect of chitosan.     

Effect of chitosan derivatives on the reproduction of coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosanN-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 inEscherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited antibacterial activity. The efficiency of inhibition of viral infection and the antibacterial activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or antibacterial activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability ofE. coli may also be due to the antibacterial effect of chitosan.     

Inactivation of coliphages by chitosan derivatives

The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and, probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan.     

Coliphages inactivation using chitosan derivatives

The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan.     

Effect of chitosan oligomer on phage particles and reproduction of phage 1-97A in Bacillus thuringiensis culture]

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensis subsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the endplate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcus flavefaciens

Highly methylated, long-chain celluloses strongly inhibited cellulose degradation by several species of cellulolytic bacteria of ruminal origin. Specifically, the inhibitory effects of methylcellulose on the growth of Ruminococcus flavefaciens FD1 were concentration dependent, with complete inhibition at 0.1% (wt/vol). However, methylcellulose did not inhibit growth on cellobiose or cellulooligosaccharides. Mixtures of methylated cellulooligosaccharides having an average degree of polymerization of 6.7 to 9.5 inhibited cellulose degradation, but those with an average degree of polymerization of 1.0 to 4.5 did not. Similar inhibitory effects by methylcellulose and, to a lesser extent, by methyl cellulooligosaccharides were observed on cellulase activity, as measured by hydrolysis of p-nitrophenyl-beta-d-cellobioside. R. flavefaciens cultures hydrolyzed cellulooligosaccharides to cellobiose and cellotriose as final end products. Cellopentaose and cellohexaose were cleaved to these end products, but cellotetraose was also formed from cellohexaose. Methylcellulose did not inhibit hydrolysis of cellulooligosaccharides. These data are consistent with the presence of separate cellulase (beta-1,4-glucanase) and cellulodextrinase activities in R. flavefaciens.     

Immunomodulating activity of chitosan derivatives with salicylic acid and its fragments

A study of biological activity of the derivatives of the chitin-chitosan oligomer with salicylic acid and its fragments showed that chitosan salicylate actively protected potato tubers against Phytophthora infestans but sharply inhibited reparation of potato tissues. N-(2-hydroxybenzyl)chitosan exhibited good protective properties but did not influence wound reparation. N-(2-hydroxy-3-methoxybenzyl)-N-pyridoxchitosan, which contained the pyridoxal and 2-hydroxy-3-methoxy fragments, was the most efficient, stimulating both defense against late blight and wound reparation in potato tissues.     

Growth and viability of mycelial fragments of white-rot fungi on some hydrogels

The viability of mycelial fragments of Trametes versicolor and Irpex lacteus and their growth on selected hydrogels are described. The size of mycelial fragments of the fungi did not significantly influence their viability. Alginate hydrogel films supported fungal growth better than agarose, carrageenan, chitosan and gelatin films, and had the highest mechanical strength but were less hydrophilic than the other hydrogels. All commercial alginates that were tested supported aseptic growth of fungal fragments without prior sterilization of the hydrogel solution. The viability of mycelial fragments in the hydrogel solutions was higher for some commercial alginates than that in laboratory grade alginate. The mechanical strength and hydrophilicity of hydrogels from alginate type Sobalg FD 155 and Meer HV were comparable to that of laboratory grade alginate. Sterilization and pH of the alginate hydrogel did not significantly influence the growth of T. versicolor mycelial fragments but affected the growth of I. lacteus. Concentrations of alginate in the range of 1–2% in the hydrogel did not affect the growth of entrapped mycelial fragments of these fungi. Received 25 June 1997/ Accepted in revised form 07 March 1998     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Antiphage properties of many kinds of chemicals such as antibiotics, surface-active agents and chelating agents were examined on Brevibacterium lactofermentum No. 2256—phage P465 system using double-layer agar method, as a part of the basic study, for preventing phage infection in the industrial fermentation.

A great majority of inhibitors which were selected were usually nonspecific and inhibited also bacterial growth. Among about 200 chemicals tested, 5 antibiotics such as chloramphenicol and tetracycline, 6 chelating agents such as phytic acid and 19 surface- active agents such as PEG monoester and POE alkyl ether showed the selective inhibitions for phage infection at the concentrations which did not affect bacterial growth, or at the subbactericidal concentrations that suppressed bacterial growth slightly.

Of the above chemicals which showed selective inhibitions for phage infection, a possible mechanism of chelating agents chiefly of phytic acid was investigated. When 0.1 to 0.2% of phytic acid was present in the medium, the effect of inhibition was most remarkable; this could be applied to the actual phage-infected l-glutamic acid fermentation. Phytic acid had no direct phagocidal action, nor did it inhibit the late step of the phage multiplication; but it prevented the adsorption of phages, which required inorganic cofactors such as Mg²⁺ or Ca²⁺ in this step, to the host bacteria. Moreover, a part of the infected bacteria was made incapable of forming plaques in the presence of phytic acid. These results suggested that the chelation between Mg²⁺ or Ca²⁺ and phytic acid would remove the metal ions essential for phage adsorption and prevent the phage adsorption and infection of phage DNA, consequently, the phage infection.

The effect of the non-ionic surface-active agents (SAA) on the infection of phage P465 of Br. lactofermentum was examined by adsorption and one-step growth experiments as a part of the basic study on the prevention of phage-infection in the industrial fermentation. Among various SAA tested, polyoxyethylene stearyl ether (POE-SE), polyethylene glycol monooleate (PEG-MO) and polyoxyethylene sorbitan monostearate (Tween 60) had remarkably demonstrated the selective inhibition of phage infection.

The effect of the above three SAA was apparently restricted to the initial adsorption step of phage infection, for the phage already adsorbed would not be affected by exposure to SAA. However, the results of one-step growth experiment indicated that Tween 60 inhibited not only the phage-adsorption, but also the maturation of phage already adsorbed in the host cells. The rate of the inhibition was found to be directly related to the concentration of agent. And, the most effective adsorption-inhibition was exhibited at the critical micelle concentration of SAA. The concentration as used in our experiments did not affect the viability of either phages or the host cells.

The results also indicated that the inhibition of phage-adsorption was due to the action of SAA on the surface of the bacterial cells rather than on the phage. This is supported by the observation that preincubation of phage with SAA did not affect either the subsequent adsorption rate or the plaque-forming ability of the phage. In contrast with above, a short-term exposure of bacterial cells to SAA caused an apparent change to the cell surface which was only partially restored by washing repeatedly. Moreover, the inhibitory effect of SAA on phage-adsorption appears quite specific in the phage-host system.     

Inhibition of Bacteriophages of Amino Acid Producing Bacteria by N-Acylamino Acids

The screening of phage inhibitors from various N-acylamino acids in the industrial amino acid fermentation was carried out, using the Brevibacterium lactofermentum No. 2256–L₁ phage system. Of the seventy-six chemicals tested, the N-acyl derivative of glutamic acid having 16 or 18 carbon atoms in N-acyl residue showed the selective inhibition for phage infection at the concentration of 20 μg/ml without affecting bacterial growth and fermentation. They were effective also on the other kinds of bacterium-phage systems. With N-palmitoyl-l-glutamic acid, its inhibitory action for phage infection was investigated. The results indicated that the chemical at the concentration of 20 μg/ml did not inhibit the phage adsorption, but diminished the number of infective center at the latent period and suppressed the number of the released phage progenies.     

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3--glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10⁶ Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.Abbreviations DP degree of polymerization - FDA fluorescein diacetate - PE pachyman equivalents     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Adhesion and growth of HUVEC endothelial cells on films of enzymatically functionalized chitosan with phenolic compounds

Human Umbilical Vein Endothelial Cell (HUVEC) growth on chitosan films and its enzymatically functionalized derivatives films with ferulic acid (FA) and ethyl ferulate (EF) was assessed by evaluating cell adhesion, morphology and cell viability. The results indicated that chitosan derivative films improved protein adsorption properties compared to chitosan films. The HUVEC cell morphology showed well attachment and spread phenotype on chitosan derivative films compared to those growing on chitosan films which did not spread and remained round. Evaluation of cell viability revealed improvement of cell adhesion on chitosan derivative films compared to chitosan film depending on the quantity of oxidized phenols grafted on chitosan. In addition, FA-/EF-chitosan films allowed almost similar cell adhesion. Furthermore, cell adhesion was increased with the film thickness. These results suggested that the oxidized phenols grafting on chitosan is a promising process to enhance cell adhesion, growth and creating useful functional biomaterials.     

Degradation of Escherichia coli B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid-Defective Amber Mutants of Bacteriophage T7

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of Escherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 10(6) between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7-induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.     

壳聚糖作为基因药物载体的研究进展

以壳聚糖及其衍生物作为基因的载体的转染效率受到许多因素的影响, 如复合物粒子大小、壳聚糖/DNA的比值、壳聚糖的分子量、脱乙酰度、转染过程中血清的浓度、介质的pH值等。对壳聚糖进行一定程度的修饰, 可以改变壳聚糖的转染效率。介绍了壳聚糖作为基因转移载体的转染条件, 转染效率和转染机制的研究情况及研究进展。     

Synthesis and bioactivities of poly(ethylene glycol)–chitosan hybrids

Poly(ethylene glycol)–chitosan hybrids of various molecular weights having different degree of substitution were synthesized, by reductive N-alkylation of chitosan with poly(ethylene glycol) aldehyde, to study their bioactivities. The influence of these chitosan derivatives on the reactive oxygen species generation from canine polymorphonuclear leukocyte cells was investigated in vitro by chemiluminescence response. Reactive oxygen species generation by the influence of poly(ethylene glycol)–chitosan hybrids was decreased with the increase of degree of substitution. The reduction of interaction of poly(ethylene glycol)–chitosan hybrids with polymorphonuclear leukocyte cells might be caused by the decrease of amino group in chitosan main chain and increase of the steric hindrance by poly(ethylene glycol) chain. The influence of the poly(ethylene glycol)–chitosan hybrids on complement component C3 activation was investigated by single radial immunodiffusion method. Influence on complement component C3 activation by poly(ethylene glycol)–chitosan hybrids was almost same as chitosan.