Preparative methods of phosphorylated chitin and chitosan--an overview.

Biomaterials such as chitin, chitosan and their derivatives have a significant and rapid development in recent years. Chitin and chitosan have become cynosure of all party because of an unusual combination of biological activities plus mechanical and physical properties. However, the applications of chitin and chitosan are limited due to its insolubility in most of the solvents. The chemical modification of chitin and chitosan are keen interest because of these modifications would not change the fundamental skeleton of chitin and chitosan but would keep the original physicochemical and biochemical properties. They would also bring new or improved properties. The chemical modification of chitin and chitosan by phosphorylation is expected to be biocompatible and is able to promote tissue regeneration. In view of rapidly growing interest in chitin and chitosan and their chemical modified derivatives, we are here focusing the recent developments on preparation of phosphorylated chitin and chitosan in different methods.

Preparation of doxorubicin-containing chitosan microspheres for transcatheter arterial chemoembolization of hepatocellular carcinoma.

A new form of doxorubicin hydrochloride (DRH)-containing chitosan microspheres (CMs) was prepared by employing an expanding-loading-shrinking (E-L-S) process. One hundred mg of pre-formed CMs were soaked in absolute ethanol and then placed in reduced pressure (the expanding process). Ten mg of DRH (2 mg ml(-1)) were added into the expanded CMs (the loading process). Next the microspheres were freeze-dried (the shrinking process). As a result of this E-L-S process, 10% (w/w) DRH-containing CMs (DRH-CM) were made. During 7 days, 22.6% of the DRH was observed to be released on the in vitro drug release study. In addition, these new DRH-CMs could be used for transcatheter arterial chemoembolization (TACE) procedure in VX2 hepatic tumour models of rabbit and the anti-tumour effects of DRH-CMs were investigated. On the post-CT scan 7 days after the TACE, total infarctions of the VX2 tumour were observed in 5 rabbits among the 6 total rabbits.

Sulfated chitin and chitosan as novel biomaterials.

Chitin and chitosan are known to be natural polymers and they are non-toxic, biodegradable and biocompatible. Chemical modification of chitin and chitosan with sulfate to generate new bifunctional materials is of interest because the modification would not change the fundamental skeleton of chitin and chitosan, would keep the original physicochemical and biochemical properties and finally would bring new or improved properties. The sulfated chitin and chitosan have a variety of applications, such as, adsorbing metal ions, drug delivery systems, blood compatibility, and antibacterial field. The purpose of this review is to take a closer look about the different synthetic methods and potential applications of sulfated chitin and chitosan. Based on current research and existing products, some new and futuristic approaches in this context area are discussed in detail. From the studies reviewed, we concluded that sulfated chitin and chitosan are promising materials for biomedical applications.

O-carboxymethyl-chitin concentration in granulocytes during bone repair.

Peroral and intravenous administrations of (14)C-labeled carboxymethyl-chitin (CM-chitin) revealed that CM-chitin accumulated in bone marrow. Thus, a composite of CM-chitin with hydroxyapatite (HA) was prepared to examine the bone repairing properties by animal and cell line experiments. The new bone formation of CM-chitin.HA composite was superior to that of CM-chitin, HA, and blank. A porous CM-chitin.HA composite is a functional material which could act as a scaffolding of osteoblast-like cells, a barrier to ingrowth of fibrous connective tissues. The cytotoxicity of CM-chitin was evaluated using the MC3T3-E1 cell line, and we found that control of degree of deacetylation is a very important factor in using CM-chitin as bone repairing material.

Mechanical properties of collagen fascicles from in situ frozen and stress-shielded rabbit patellar tendons.

OBJECTIVE: To know the effects of stress shielding on the biomechanical properties of collagen fascicles obtained from in situ frozen patellar tendons (an autograft model). DESIGN: Collagen fascicles of approximately 300 microm in diameter were obtained from in situ frozen rabbit patellar tendons and also from in situ frozen and stress-shielded ones, and their mechanical properties and fibroblast density were determined. BACKGROUND: Stress shielding changes the mechanical properties of in situ frozen patellar tendons in which there exist no fibroblasts. The mechanisms of this phenomenon have not been studied well. METHOD: Patellar tendons of both in situ frozen group and in situ frozen and stress-shielded group were frozen in situ by liquid nitrogen to kill fibroblasts. Then, in the in situ frozen and stress-shielded group, no tension was applied to the tendons for 2, 3, and 6 weeks, while normal tension was applied to the tendons of the in situ frozen group. Tensile properties of the collagen fascicles obtained from these tendons were determined using a microtensile tester, and were compared to the collagen fascicles from non-frozen, stress-shielded patellar tendons. RESULTS: Tangent modulus and tensile strength of collagen fascicles from the in situ frozen and stress-shielded group progressively decreased with the time of stress shielding; however, these decreases were much smaller than those of the fascicles obtained from non-frozen, stress-shielded tendons. Although there were few fibroblasts in the patellar tendon of the in situ frozen and stress-shielded group at 2 weeks, the modulus and strength of the fascicles from the posterior portion were significantly lower than those in the in situ frozen group. In addition, the reduction of strength caused by stress shielding was much smaller in collagen fascicles than in bulk patellar tendons. CONCLUSION: The mechanical properties of collagen fascicles in in situ frozen tendons (an autograft model) are affected by stress shielding even under acellular condition. RelevanceThe in situ frozen, stress-shielded patellar tendon is a model of augmented autografts which are clinically used for the reconstruction of injured anterior cruciate ligaments. The sub-macroscopic studies of the tendon are useful to understand the mechanisms of the reduction of graft strength and its gradual recovery observed after reconstruction.

Immunological aspects of chitin and chitin derivatives administered to animals.

Chitosan amino groups are recognized by the immune system. Therefore, every derivative of chitin should be assayed immunologically if biomedical applications are sought. Macrophages are activated to various extents by chitin derivatives. Deacetylated chitin (30% deacetylation) and chitin sulfate stimulate the production of circulating antibodies. Accumulation of carboxymethyl chitin takes place in granulocytes and macrophages. These polysaccharides activate complement in analogy to zymosan. Intraperitoneal injection of N-acetylchitohexaose inhibits the growth of tumor cells and pathogens on a similar level as that of lentinan.

[Adaptation of dental home care--report number 1: Limitation of dental home care].

In 1985, we established a home care team which treats elderly and handicapped patients who cannot easily come to our clinic for treatments. The demand for home care is increasing, and 70% of this demand is related to dentures-i.e., adjusting dentures or making new dentures. Through the use of portable equipment, the home care team is able to cope effectively with this demand. However, some patients who experience difficulty eating as a result of their dental conditions require immediate attention. Furthermore, some patients require frequent follow-up. In such cases, time becomes more of an issue. Cases which were found to be difficult for the home care team include: treatments involving existing teeth where more than one tooth was involved, severe cares requiring surgical treatment, and cases requiring close monitoring of a patient's physical conditions. From our experience we know there is a great demand for home care, especially among elderly and handicapped patients; we also know that the risk factors increase and that there are limitations to the kind of care we can effectively deliver. Therefore in order to ensure the patient's safety and conduct effective treatment, it is critical that we evaluate each patient's condition carefully and thoroughly. Furthermore, it is important to develop a treatment plan and to conduct treatment while comprehensively monitoring the patient's condition.

[Adaptation of dental home care--report number 2: Attempt at a new system of dental home care].

In the first report we revealed some problems in visiting dental treatment, and concluded that a new dental care system should be constructed for the solution of those problems. Therefore, we started a new system which included dental treatment under hospitalization in our dental hospital. For home patients this system aims at treating, managing smoothly and providing better dental treatment, due to the choice of hospitalization, outpatient care, or home visits in the medical process. In the period from March, 1993, when our dental hospital was established, until December, 1998 treatment was given 1,527 times with 420 patients under this system, and 127 of these patients chose dental treatment under hospitalization. Dental treatment under hospitalization is the management method not found in usual dental treatment, but it is indispensable to our system. When we decide hospitalization, we must make an overall estimate of the patient's general condition, contents of treatment, eating function and nutritional condition, background, and the wishes of the patient and family. In principle, visiting dental treatment is intended for a patient who has finished dental treatment. When treatment is necessary, it should be limited to simple treatment, first aid and maintenance. The oral care of many home patients under the present circumstances is not practiced sufficiently, and cooperation of medical and welfare workers is required to improve such conditions.

Endothelial cell responses to chitin and its derivatives.

The effects of chitin and its derivatives on the proliferation of human umbilical vein endothelial cells (HUVECs) and on the production of cytokines were examined in vitro. Chitin and its derivatives had no effect on the proliferation of cultured HUVECs. N-Sulfonated 70% deacetylated chitin (S-DAC70) stimulated the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha from HUVECs. Compared to S-DAC70, the other materials tested in the present study showed less effect in the stimulation of IL-8 and TNF-alpha production and had no effect in the stimulation of IL-1beta and IL-6 production. These results indicated that S-DAC70 affects HUVECs function but not proliferation.

Regioselective syntheses of sulfated polysaccharides: specific anti-HIV-1 activity of novel chitin sulfates.

A novel and convenient method for the regioselective syntheses of sulfated analogs of chitin and chitosan is described in relation to studies on structure-biological activity. Fully protected, soluble derivatives of chitosan were found to be useful intermediates for the syntheses of a novel class of sulfated polysaccharides, 2-acetamido-2-deoxy-3-O-sulfo-(1-->4)-beta-D-glucopyranan (3-sulfate, 3S, 4) and (1-->4)-2-deoxy-2-sulfoamido-3-O-sulfo-(1-->4)-beta-D-glucopyranan (2,3-disulfate, 23-S, 3). These compounds were tested for their activities in (i) inhibiting HIV-1 replication in vitro and (ii) inhibiting blood coagulation. The results reveal that the selective sulfation at O-2 and/or O-3 affords potent antiretroviral agents showing a much higher inhibitory effect on the infection of AIDS virus in vitro than that by the known 6-O-sulfated derivative (6-sulfate, 6S). Moreover, the 23-S product completely inhibited the infection of AIDS virus to T lymphocytes at concentrations as low as 0.28 microgram/mL without significant cytotoxicity. The regioselective introduction of sulfate group(s) at O-2 and/or O-3 had little effect on generating anticoagulant activity, whereas 6-O-sulfated chitin strongly inhibits blood coagulation. These results suggest that the specific interaction of these new types of chitin sulfates with gp 120 of the AIDS virus depends significantly on the sites of sulfation rather than on the total degree of substitution on sugar residues.

Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro.

The effects of chitin and its derivatives on the proliferation of fibroblasts and on the production of cytokines were examined in vitro. Chitin and its derivatives showed almost no acceleratory effect on the proliferation of cultured fibroblasts. On the contrary, high-concentration 500 micrograms ml-1) D-glucosamine cultures supplemented with or without a 10% fetal calf serum (FCS) supplementation showed a significant (P < 0.05) reduction in the rate of proliferation of L929 fibroblast cells relative to control. High-concentration chitosan cultures supplemented with 10% FCS showed a significant (P < 0.05) reduction in the rate of L929 fibroblast proliferation. However, the inhibition of cell proliferation by high concentrations of chitosan did not show in cultures without FCS. Interleukin-8 (IL-8) was induced in the supernatants of rat primary cultured dermal fibroblasts stimulated with chitin and its derivatives. Chitin and its derivatives did not stimulate the production of IL-6 by mouse dermal primary cultured fibroblasts. IL-1 alpha, IL-1 beta and tumour necrosis factor-alpha were not detected in the fibroblast supernatants. These observations support the notion that cell proliferation is accelerated indirectly by chitin and its derivatives when these materials are used in vivo. In vivo findings of a angiogenesis and migration of neutrophils may be due to persistent release of IL-8 from fibroblasts.

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

Marked effect of DNA on collagen fibrillogenesis in vitro.

Growth of collagen fibrils in the presence of DNA was more rapid than that in the absence of DNA. Some of collagen fibrils formed in the presence of DNA were significantly wider than those in the absence of DNA. Moreover, the cross-bandings were also very distinct in spite of using pepsin-digested collagen. These results suggest that DNA not only adsorbs to collagen but induces the extraordinary fibrillogenesis of collagen.

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme.

Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.

DNA-alginate complex recognized by autoantibodies against DNA.

Double-stranded DNA was effectively complexed with alginic acid and immobilized on a surface of polystyrene microtiter plate. Dose-dependent binding of anti-DNA autoantibodies was finely observed to the solid phase DNA-alginate complex in enzyme-linked immunosorbent assay (ELISA). In contrast, non-specific binding of antibodies to alginate was scarcely detected rather than to poly-L-lysine. These results shown an availability of the solid phase DNA-alginate complex as an antigen in ELISA for detection of anti-DNA antibodies.

Interglycosidic acetals. Part 3. Synthesis and structure determination of cyclic monobenzylidene acetals of cyclodextrin derivatives bridging between two contiguous D-glucopyranosyl residues.

Transacetalation of fully 6-O-pivaloylated alpha-, beta-, and gamma-cyclodextrins with benzaldehyde dimethylacetal in the presence of (+)-10-camphorsulfonic acid gave monobenzylidene acetals (4, 5, 6) in moderately good yields. Benzylation of the beta-cyclodextrin derivative 5 followed by acid-catalyzed hydrolysis of the benzylidene group and acetylation afforded a di-O-acetyl-non-adeca-O-benzyl derivative 9. NMR spectroscopic analysis of 9, including two-dimensional HOHAHA and 1H-(13)C correlation experiments revealed that the benzylidene group bridged the O-2 and O-3 positions of contiguous D-glucopyranosyl residues. Reductive ring-opening of the benzylidene acetal with lithium aluminum hydride/aluminum chloride afforded predominantly a 2(1)-O-unprotected derivative 10 in good yield.

Synthetic glycogels for affinity electrophoresis: a facile and efficient method for investigating sugar-lectin interaction.

A novel and efficient method for analyzing sugar-lectin interaction using affinity electrophoresis (AEP) is described. Polyacrylamide gels covalently conjugated with 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) residues were successfully prepared by radical copolymerization of highly reactive 3-(N-acryloylamino)propyl 2-acetamido-2-deoxy-beta-D-glucopyranoside with acrylamide in the presence of N,N'-methylenebisacrylamide (BIS). When the glycogels carrying various densities of GlcNAc branches were employed for polyacrylamide gel electrophoresis (PAGE) of lectins, the mobilities of wheat germ agglutinin (WGA) were specifically reduced by increasing the concentrations of the GlcNAc residues in gels, although concanavalin A (Con A) showed no significant change in the mobility. It was also demonstrated that the association constant of WGA with immobilized GlcNAc residue can be determined by combined use of this stable glycogel and an automated gel-scanning system associated with fluorometric spectroscopy. The association constant of WGA with the GlcNAc moiety was estimated to be 1.24 x 10(4) M-1.

Utilization of DNA as functional materials: preparation of filters containing DNA insolubilized with alginic acid gel.

Thin films composed of DNA and alginic acid were prepared by casting their mixed solution on glass plate followed by coagulation with aqueous solution of calcium chloride. DNA could be conveniently insolubilized by this method. DNA in the films adsorbed intercalating materials, such as ethidium bromide. This phenomenon was successfully applied to the preparation of filters for the selective removal or accumulation of harmful intercalating pollutants.

A sulfated chitin, SCM-chitin III, inhibits the clearance of human erythrocytes from the blood circulation in erythrocyte-transfused SCID mice.

Recently, we have developed a SCID mouse model in which circulating red blood cells (RBC) are entirely substituted with RBCs from other animals like bovine (Bo) or human (Hu). The relatively short life time, especially of Hu-RBCs, in the SCID mouse, however, is a major obstacle in this model. The present study was performed to examine whether a low-toxic sulfated chitin, carboxymethyl chitin III (SCM-chitin III), which has heparin-like structures in the molecule (heparinoid), could inhibit the Hu-RBC clearance in RBC-transfused SCID mice. When Hu-RBCs were transfused simultaneously with SCM-chitin III, their life time in the blood circulation was prolonged significantly. Sulfated chitosan (S-chitosan) showed only a weak decelerating activity on the clearance of Hu-RBCs. Carboxymethyl chitin (CM-chitin), which was used as an unsulfated control compound, had no effect on the Hu-RBC clearance. Another sulfated polysaccharide, dextran sulfate, though this showed some adverse effects, such as anti-coagulant and anti-platelet aggregation, also exhibited a potent decelerating activity on Hu-RBC clearance. Clearance deceleration by these sulfated polysaccharides was primarily attributable to the inhibition of RBC uptake by cultured macrophages.

Synthesis and conformational investigation of tandem repeat sequence in RNA polymerase II.

The largest subunit of RNA polymerase II has a very interesting sequence in the C-terminus; that is, a tandem repeat sequence of Ser-Pro-Thr-Ser-Pro-Ser-Tyr consisted of proline residues and three kinds of residues having side-chain hydroxyl groups. Although lack of this tandem repeat is a lethal event in vivo, its functional role is unclear. The sequential polypeptide corresponding to this tandem repeat, poly(Ser-Pro-Thr-Ser-Pro-Ser-Tyr), was synthesized and its conformation was investigated by circular dichroism comparing to the monomeric heptapeptide. In addition, the theoretical conformational analysis based on the molecular mechanics was tried for the heptapeptide in the repeating unit and the periodic polyheptapeptide corresponding to the tandem repeat sequence. These results suggested the possibility that the tandem repeat contains a kind of super conformation composed of the repetitive turn structure in the native state. The characteristic repetitive turn structure would be the key of its function mechanism.