Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation

The functional properties of three shrimp chitosan preparations with different degrees of deacetylation (75%, 87% and 96% DD) but with a constant molecular weight (about 810 kDa) were investigated. Chitosan with 75% DD had a 1.5 times higher water absorption, probably due to its 20% lower level of crystallinity. Membranes cast from this chitosan also exhibited 1.5 times more water absorption and 2 times higher permeability. However, chitosan with 87% and 96% DD had 1.5-2 times higher absorption of fat and the orange II dye. This is attributed to the higher content of positively charged amine groups in the polymer. Cast into membrane, chitosan of higher degree of deacetylation showed a higher tensile strength and a higher elongation at break, probably due to the higher level of crystallinity.     

Prevalence of human alveolar echinococcosis in Hokkaido as evaluated by western blotting

The Western blotting (WB) method was used to evaluate the prevalence of human alveolar echinococcosis in Hokkaido, Japan. One hundred and sixteen WB-positive serum specimens were found in 1987 and 1988, and were grouped into two serologically different types from their WB-immunostaining patterns: "complete type", that showed multiple bands with various molecular weights ranging from 29 to 205 kilodaltons (kDa), and "incomplete type", that revealed a few bands with low molecular weights of 30-35 kDa and/or with molecular weights higher than 90 kDa. Therefore, we found that the WB method is applicable for the sero-epidemiological studies of human alveolar echinococcosis in Hokkaido.     

Effect of ultrasonic treatment on the biochemphysical properties of chitosan

Four chitosans with different molecular weights and degrees of deacetylation degree and 28 chitosans derived from these initial chitosans by ultrasonic degradation have been characterized by gel permeation chromatography (GPC), FT-IR spectroscopy, X-ray diffraction and titrimetric analyses. Antimicrobial activities were investigated against E. coli and S. aureus using an inhibitory rate technique. The results showed that ultrasonic treatment decreased the molecular weight of chitosan, and that chitosan with higher molecular weight and higher DD was more easily degraded. The polydispersity decreased with ultrasonic treatment time, which was in linear relationship with the decrease of molecular weight. Ultrasonic degradation changed the DD of initial chitosan with a lower DD (<90%), but not the DD of the initials chitosan with a higher DD (>90%). The increased crystallinity of ultrasonically treated chitosan indicated that ultrasonic treatment changed the physical structure of chitosan, mainly due to the decrease of molecular weight. Ultrasonic treatment enhanced the antimicrobial activity of chitosan, mainly due to the decrease of molecular weight.     

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC₅₀ value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K_i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.     

Reactive oxygen species scavenging activity of aminoderivatized chitosan with different degree of deacetylation

Chitosans with different degree of deacetylation were prepared from crab shell chitin in the presence of alkali. Aminoderivatized chitosan derivatives were prepared in addition of amino functional groups at a hydroxyl site in the chitosan backbone. Six kinds of aminoderivatized chitosan such as aminoethyl-chitosan (AEC90), dimethylaminoethyl-chitosan (DMAEC90), and diethylaminoethyl-chitosan (DEAEC90), which were prepared from 90% deacetylated chitosan, and AEC50, DMAEC50 and DEAEC50, which were prepared from 50% deacetylated chitosan, were prepared and their reactive oxygen species (ROS) scavenging activities were investigated against hydroxyl radical, superoxide anion radical and hydrogen peroxide. The electron spin resonance (ESR) spectrum revealed that AEC90 showed the highest scavenging effects against hydroxyl and superoxide anion radical, the effects were 91.67% and 65.34% at 0.25 and 5 mg/mL, respectively. For hydrogen peroxide scavenging effect, DEAEC90 exhibited the strongest activity. These results suggest that the scavenging effect depends on their degree of deacetylation and substituted group.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

Antihypertensive activity of chitin derivatives

To develop angiotensin I converting enzyme (ACE) inhibitory chitin derivatives based on the properties of ACE inhibitors, chitins with different degree of deacetylation were chemically modified by grafting 2-chloroethylamino hydrochloride onto chitin at the C-6 position. Three kinds of chitin derivatives were prepared and designated as aminoethyl-chitin (AEC) with 10% degree of deacetylation, aminoethyl-chitin with 50% degree of deacetylation (AEC50), and aminoethyl-chitin with 90% degree of deacetylation (AEC90). IC50 values of three chitin derivatives on ACE were 0.064 microM (AEC), 0.038 microM (AEC50), and 0.103 microM (AEC90). The results of Dixon plots revealed that AEC50 was a competitive inhibitor, and the inhibition constant (Ki) value was 0.021 microM. In addition, the antihypertensive effect of AEC50 on spontaneously hypertensive rats (SHR) was evaluated, and the result showed that it effectively decreased systolic blood pressure (SBP) in a dose-dependent manner.     

Deacetylation of chitin under homogeneity

Chitin isolated enzymatically from Antarctic krill shells was dissolved in aqueous NaOH by freezing and thawing to create homogenous conditions. Deacetylation was performed at room temperature or heating. The degree of deacetylation, molecular weight, and dynamic viscosity of solutions were estimated in chitosan samples. Deacetylation of chitin under homogenous conditions was optimized. Chitosans with molecular weights of 180-220 and 250-300 kDa were obtained from the chitins of Antarctic krill and Northern shrimps, respectively.     

Bioengineering functional copolymers. XXI. Synthesis of a novel end carboxyl-trithiocarbonate functionalized poly(maleic anhydride) and its interaction with cancer cells

A novel carboxyl-trithiocarbonate functionalized polymer with a highly selective antitumor activity was synthesized by a reversible addition-fragmentation chain transfer (RAFT) polymerization of maleic anhydride (MA) with benzoyl peroxide as an initiator and S-1-dodecyl-S-(α,α'-dimethyl-α″-acetic acid)trithiocarbonate as a RAFT agent with the aim to design and synthesize an effective anticancer agent with minimum side effects. The structure, molecular weights and composition of synthesized polymers were investigated by (1)H ((13)C) NMR, MALDI-TOF-MS and GPC analyzes. It was demonstrated that RAFT polymerization of MA was accompanied by a partially controlled decarboxylation of anhydride units and the formation of conjugated double bond fragments in backbone macromolecular chains. The mechanism of interaction of pristine RAFT agent and PMA-RAFT polymer with cancer (HeLa human cervix carcinoma) and normal (L929 Fibroblast) cells was investigated by using a combination of chemical, biochemical, statistical, spectroscopic (SEM and fluorescence inverted microscope) and real-time analysis (RTCA) methods. PMA-RAFT exhibited higher and selective cytotoxicity, apoptotic and necrotic effects toward HeLa cells at relatively low concentrations (around 7.5-75μgmL(-1), IC(50)=11.183μgmL(-1)) and toward Fibroblast cells at high concentrations (IC(50)>100μgmL(-1)). The observed highly selective antitumor activity render PMA-RAFT polymers as promising candidates for the utilization in cancer chemotherapy.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Renin inhibition activity by chitooligosaccharides

Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity.     

Aberrant mobility phenomena of the DNA repair protein XPA

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from approximately 40-45 kDa) and gel filtration chromatography ( approximately 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a approximately 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA +/- mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from approximately 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.     

Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.     

来源于家蝇、地鳖虫、黄粉虫和河虾壳几丁糖的基本特性

为了了解昆虫来源几丁糖的基本特性及其与河虾来源几丁糖的差别,用同一方法在相同条件下分别以河虾壳(shell of Procambarus clarkii)、家蝇蛹壳(pupa shell ofMusca domestica vicinaMacquart)、地鳖虫壳(shell of Euplyphaga Walker)和黄粉虫蜕(exuviate of Tnebrio molitorL.)为来源制备的几丁糖在灰份、脱乙酰度、分子质量等及红外图谱上进行比较研究。昆虫来源几丁糖,其灰份低于河虾壳来源几丁糖;家蝇蛹壳几丁糖分子质量明显低于其他3种来源的几丁糖;从几丁糖质量角度看,采用家蝇蛹壳和地鳖虫壳可制备脱乙酰度较高的几丁糖;4种来源的几丁糖红外光谱图谱基本一致,具有几丁糖的特征吸收峰。     

Preparation of chitosan microparticles and study of their interaction with interferon

Chitosan with viscosity average molecular weights of 340, 281, 199, 137, and 42 kDa was used to prepare microparticles by precipitation coacervation. Acid and enzymatic hydrolysis yielded chitosan samples with a deacetylation degree of 0.85 ± 0.03. The chitosan microparticles were 0.85–1.7 μm in size and had a ζ-potential of 30.7?38.6±0.1 mV. Testing the microparticles for toxicity showed that they caused no death of animals. Interaction of recombinant α-2 interferon with the microparticles was studied by sorption from solutions. The maximum adsorption efficiency of interferon was 88% and the capacity of microparticles was 11.8–12.7 μg/mg.     

Effects of molecular weight and hydrolysis conditions on anticancer activity of fucoidans from sporophyll of Undaria pinnatifida

Hydrolyzed fucoidans, from sporophyll of Undaria pinnatifida, were used to determine the effects of molecular weight (Mw) and hydrolysis conditions on cancer cell growth. Native fucoidans showed anticancer activity of 37.6%. When hydrolyzed in boiling water with HCl for 5 min, fucoidans (Mw = 490 kDa) significantly increased anticancer activity to 75.9%. However, fucoidans hydrolyzed in a microwave oven showed little improvement of anticancer activity and even exhibited the inhibition activity below 30% when treated more than 90s. This suggests that anticancer activity of fucoidans could be significantly enhanced by lowering their Mw only when they are depolymerized by mild condition.     

Emulsion stabilizing properties of various chitosans in the presence of whey protein isolate

The emulsion stabilizing potential of chitosans (CN) with various molecular weights and degrees of deacetylation (CNI=1494 kDa, 78.1%DD; CNHI=694 kDa, 78.5%DD; CNHI2=319 kDa, 78.5%DD; CNHK=749 kDa, 67.7%DD) was compared in the presence of whey protein isolate. Phase separation evolutions revealed minimal stabilizing concentrations against syneresis from 0.1 to 0.125% in most CN preparations, except CNHI2 where it was higher (0.225%). Those stabilizing concentrations are the result of interfacial coadsorption saturation of CN with proteins, favouring interfacial electrostatic and steric stabilizing mechanisms. The emulsion characteristics (droplet size, limiting low-shear viscosity, and surface net charge), mostly distinctive at 0.1%CN, revealed the following order of stabilizing potentials: CNI≈CNHI>CNHK>CNHI2, which is in agreement with respective phase separation evolutions. The lower stabilizing potential of CNHI2 is explained by lower interfacial coadsorption efficiency with protein. In spite of a higher interfacial load of CNHK vs. CNI and CNHI, its lower stabilizing potential is essentially explained by a lower surface net charge.     

Quantification and characterization of insoluble chitinous materials in viscous chitosan solutions

Insoluble chitinous materials in highly viscous chitosan solutions can be quantified using the viscosity-lowering action of transglucosidase (EC 2.4.1.24). In chitosan, commonly produced by high temperature deacetylation (90 °C), between 70–90% of insoluble chitinous materials were recovered by this enzymatic method whereas only 25% recovery was obtained by the nitrous acid method. The insoluble material recovered after enzyme treatment had a higher degree of deacetylation and a lower degree of crystallization than that after nitrous acid treatment. The results are explained by difference in penetration by enzyme and nitrous acid into the insoluble particle.     

Polymersome encapsulated hemoglobin: a novel type of oxygen carrier

Bovine hemoglobin (Hb) was encapsulated inside polymer vesicles (polymersomes) to form polymersome encapsulated Hb (PEH) dispersions. PEH particles are 100% surface PEGylated with longer PEG chains and possess thicker hydrophobic membranes as compared to conventional liposomes. Polymersomes were self-assembled from poly(butadiene)-poly(ethylene glycol) (PBD-PEO) amphiphilic diblock copolymers with PBD-PEO molecular weights of 22-12.6, 5-2.3, 2.5-1.3, and 1.8-0.9 kDa. The first two diblock copolymers possessed linear hydrophobic PBD blocks, while the later possessed branched PBD blocks. PEH dispersions were extruded through 100 and 200 nm pore radii membranes. The size distribution, Hb encapsulation efficiency, P(50), cooperativity coefficient, and methemoglobin (metHb) level of PEH dispersions were consistent with values required for efficient oxygen delivery in the systemic circulation. The influence of different molecular weight diblock copolymers on the physical properties of PEH dispersions was analyzed. PBD-PEO copolymers with molecular weights of 22-12.6 and 2.5-1.3 kDa completely dissolved in aqueous solution to form polymersomes, while the other two copolymers formed a mixture of solid copolymer precipitates and polymersomes. PEHs self-assembled from 22-12.6 and 2.5-1.3 kDa PBD-PEO copolymers possessed Hb loading capacities greater than PEG-LEHs, PEGylated actin-containing LEHs, and nonmodified LEHs, although their sizes were smaller and their hydrophobic membranes were thicker. The Hb loading capacities of these polymersomes were also higher than lipogel encapsulated hemoglobin particles and nanoscale hydrogel encapsulated hemoglobin particles. PEH dispersions exhibited average radii larger than 50 nm and exhibited oxygen affinities comparable to human erythrocytes. Polymersomes did not induce Hb oxidation. The interaction between Hb and the membrane of 2.5-1.3 kDa PBD-PEO polymersomes improved the monodispersity of these particular PEH dispersions. These results suggest that PEHs could serve as efficient oxygen therapeutics.