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Abstract  Aqueous purified extract (PE) was isolated from the crude extract (CE) of the marine mollusk, Ruditapes philippinarum. Two water-soluble polysaccharides, PEF1 and PEF2, were purified from PE. The chemical structures were determined using FTIR, GC, HPLC and 13C NMR. The results indicated that PEF1 and PEF2 were homoglucan–protein complexes with a protein content of 26.0 and 8.2%, and their average molecular weights were about 2.0 × 106 and 5.0 × 103, respectively. The glucan moiety of PEF1 was mainly a (1 → 6)-branched (1 → 4)-α-d-glucan, while that of PEF2 contained (1 → 4)-α-d-glucan and (1 → 6)-β-d-glucan. PE fractions contained large quantities of glutamic acid and aspartic acid, but no cysteine. The antitumor activities were tested both in vitro and in vivo. PE showed significantly the highest tumoricidal activity against human hepatoma SMMC-7721 cells, and PEF1 had better cytotoxicity than PEF2. There was no observed cytotoxicity in human hepatocyte HL-7702 cells within the experimental concentration range for all PE fractions. PE showed antitumor activity against solid tumor Sarcoma 180 in a dose-dependent manner. It also demonstrated stimulating effect on murine lymphocyte proliferation induced by concanavalin A. The results demonstrated that the presence of bound protein, compositional glucan and moderate molecular mass would be helpful to the enhancement of antitumor activities. This study suggested that the polysaccharides isolated from the marine mollusk had a potential application as natural antitumor and immunomodulator agents. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the present study was to investigate the antioxidant activity and immunostimulating property of glucosamine (GlcN) using various in vitro and in vivo tests. Results showed that GlcN possessed excellent antioxidant activities as manifested by strong chelating effect on ferrous ions and protection of macromolecules such as protein, lipid, and deoxyribose from oxidative damage induced by hydroxyl radicals. The immunostimulating effects of GlcN were further evaluated through various immunological tests. GlcN showed excellent activity of enhancing splenocyte proliferation. Neutral red pinocytosis and NO production in mouse peritoneal macrophages were significantly augmented. Oral administration of GlcN to mice for 20 days significantly enhanced the serum antibody level in mice in response to sheep red blood cells (SRBC), increased the relative organ weight of spleen and thymus tissue, and promoted the delayed-type hypersensitivity (DTH) against SRBC as compared with control group. In conclusion, the present investigation reveals GlcN is biologically functional in antioxidative activities and immunostimulating properties. [Copyright &y& Elsevier]

Abstract: The protective effects of chitosan oligosaccharide (COS), d-glucosamine (GlcNH2) and N-acetyl-d-glucosamine (GlcNAc) on carbon tetrachloride (CCl4)-induced hepatotoxicity and the possible mechanisms that involved were investigated in male ICR mice. CCl4 (20mg/kg body weight, i.p.) administration induced marked increase in serum AST and ALT activities, primed liver lipid peroxidation, depleted sulfhydryl content, impaired total antioxidant capabilities and induced genotoxicity 24h after administration. Pretreatment with COS, GlcNH2, and GlcNAc (1.5g/kg body weight, i.g.) for 12 consecutive days prior to CCl4 challenge significantly induced metallothionein (MT) expression. Thus, the antioxidant defensive system in the body was strengthened to counteract the oxidative damage induced by the succedent CCl4 administration. Serum AST and ALT activities were effectively decreased. Hepatic malondialdehyde formation was inhibited and sulfhydryl contents, total antioxidant capabilities were markedly restored. Genotoxicity as reflected by DNA fragmentation, however, was not mitigated by pretreatment with COS, GlcNH2, and GlcNAc. Histophathologic results of liver also confirmed their hepato-protective effects. Pretreatment with COS, GlcNH2, and GlcNAc also could significantly decrease serum creatinine and uric acid levels and inhibit lipid peroxidation in kidney homogenate. Our results suggest that pretreatment with COS, GlcNH2, and GlcNAc can efficiently protect mice against CCl4-induced toxicity. [Copyright &y& Elsevier]

Catalase from Bacillus sp. N2a (BNC) isolated from Antarctic seawater was purified to homogeneity. BNC has a molecular mass of about 230 kDa and is composed of four identical subunits of 56 kDa. The catalase showed optimal activity at 25 °C and at a pH range of 6-11. The enzyme could be inhibited by azide, hydroxylamine, and mercaptoethanol. These characteristics suggested that BNC is a small-subunit monofunctional catalase. The activation energy of BNC was 13 kJ/mol and the apparent kcat/Km values were 3.6 × 106 and 4 × 106 L·mol-1·s-1 at 4 and 25 °C, respectively. High catalytic efficiency of BNC at low temperatures enables this bacterium to scavenge H2O2 efficiently. BNC exhibited activation energy, catalytic efficiency, and thermostability comparable with some mesophilic homologues. Such similarity of enzymatic characteristics to mesophilic homologues, although uncommon among the cold-adapted enzymes in general, has also been observed in other psychrophilic small-subunit monofunctional catalases. La catalase de Bacillus sp. N2a (BNC) isolée des eaux de l’Antarctique a été purifiée à homogénéité. La BNC a une masse moléculaire d’environ 230 kDa et elle est composée de quatre sous-unités identiques de 56 kDa. La catalase démontrait une activité maximale à 25 °C et dans une gamme de pH de 6 à 11. L’enzyme pouvait être inhibée par l’azide, l’hydroxylamine et le mercaptoéthanol. Ces caractéristiques suggèrent que la BNC soit une catalase monofonctionnelle à petites sous-unités. L’énergie d’activation de la BNC était de 13 kJ/mol et le kcat/Km apparent était de 3,6 × 106 et 4 × 106 L·mol-1·s-1 à 4 et 25 °C, respectivement. Le haut rendement catalytique de la BNC à basse température permet à cette bactérie de piéger efficacement le H2O2. La BNC montrait une énergie d’activation, un rendement catalytique et une thermostabilité comparable à celles d’homologues mésophiles. Une telle similarité des caractéristiques enzymatiques avec celles d’homologues mésophiles, quoique peu commune chez les enzymes adaptées au froid en général, a aussi été observée chez d’autres catalases monofonctionnelles à petites sous-unités des psychrophiles. [ABSTRACT FROM AUTHOR]

On the basis of previous achievements in Porphyra seedling production using enzymatically isolated thallus cells, we have investigated the influences of water content of thallus before freezing, storing temperature and enzymes on the viability of isolated cells. We have also tried outdoor cultivation of seedlings produced enzymatically in different seasons. It has been found that survival rate of isolated cells of thallus stored for two months can reach 90% if water content is 30% before freezing and storing temperature is constant at -20 °C. The 80% cell survival rate can be reached after two years of storage under this condition. The fluctuation of storing temperature causes drastic decrease of cell survival rate. 84% of the cells have survived digestion of sea snail enzyme I, which is the highest among five enzymes used. Outdoor cultivation of seedlings produced on the first ten days of January can reach lengths of more than 20 cm upon harvesting in the middle of May. In addition, seedlings produced in spring can be stored frozen and cultivated in autumn. The harvesting date will advance 15 days in comparison with the conventional seedling producing approach. This promises multiple rounds of cultivation of Porphyra within a year. % [ABSTRACT FROM AUTHOR]

Postmenopausal osteoporosis has seriously affected the life quality of elderly women. A natural polymer, chitin, obtained from shells of crab and shrimp, has been widely used in the biomedical field owing to its nontoxicity, biocompatibility, and biodegradability. In this study, natural N-acetyl-D-glucosamine (NAG) was prepared from liquefied chitin. The protective activities of NAG in postmenopausal osteoporosis were evaluated on Sprague Dawley rats and osteoblast-based models. Results showed that oral administration of NAG boosted trabecular bone volume and trabecular numbers. Additionally, the calcium content in the femur and tibia increased, and femoral biomechanical properties improved. Furthermore, NAG supplementation significantly lowered alkaline phosphatase levels and increased calcium content in the serum of ovariectomized rats. In vitro studies showed that NAG markedly promoted cell proliferation and stimulated osteoblast differentiation of mouse calvaria origin MC3T3-E1 cells with increased alkaline phosphatase activity in a concentration-dependent manner. Moreover, NAG effectively protected osteoblasts from oxidative damage induced by hydrogen peroxide. In conclusion, our data provide an additional foundation for dietary supplementation of NAG, which could protect and reverse osteopenia in postmenopausal women. [ABSTRACT FROM AUTHOR]

Chitosanase is able to specifically cleave ß-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase- substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substratebinding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the potential usage of biological delivery membranes containing mitomycin C (MMC) or 5-fluorouracil (5-FU) in the construction of glaucoma-filtering blebs, and to evaluate their safety and efficacy. Methods: Chitosan was selected as the biological membrane carrier to prepare sustained-released membranes. Twelve micrograms of 5-FU or MMC was covalently conjugated onto the membranes by solvent volatilization. Rabbits underwent glaucoma filtration surgery and were randomly allocated into one of the four treatment regimens: glaucoma filtration operation with no implantation of chitosan membrane group (as control), drug-free chitosan membrane implantation group (blank/placebo group), membrane containing 5-FU treatment group (5-FU group), and membrane containing MMC treatment group (MMC group). Each group consisted of 12 rabbits. Intraocular pressure (IOP) was measured and evaluated over a 28-day period follow-up preoperatively, then after surgery on days 1, 3, 5, 7, 14, 21, and 28 by Tono-Pen. The aqueous humor was analyzed in each experimental and control groups at days 4, 6, 8, 10, 12, 14, 16, and 20 after operation. Bleb survival and anterior segment were examined with a slit lamp microscope and photographed simultaneously. Two rabbits from each group were killed on day 28 and eight eye samples obtained for histopathological study. Corneas and lenses were examined by transmission and scanning electron microscopy. Results: Both 5-FU and MMC significantly prolonged bleb survival compared with control groups. The filtering bleb's survival period was significantly more prolonged in the MMC and 5-FU groups (maintained 14 days) than the other two groups (maintained 7 days). Significantly lower IOP was observed within the control, blank, and 5-FU groups after surgery on day 14 compared with that before operation, with F-values of 6.567, 11.426, and 13.467, respectively (P < 0.01). The most significant lower IOP was recorded in the MMC group on day 28 postoperation (F-value 26.866, P < 0.01). No obvious abnormalities were found in cornea or anterior lens capsule 28 days after surgery. Conclusion: The study provided evidence that 5-FU and MMC biological delivery membranes could significantly improve the outcome of filtering procedures, the survival of the bleb, and maintenance of lower IOP. MMC membrane is superior to 5-FU, with regard to the more effective reduction of IOP. The results indicated a safe and effective treatment strategy in glaucoma surgery. [ABSTRACT FROM AUTHOR]

Carboxymethyl chitosan (CM-chitosan), which is a water-soluble derivative of chitosan, has attracted much attention as a new biomedical material. The safety study of this material was persuasive for its potential application. The present study was conducted to assess the tissue distribution, pharmacokinetics, biodegradation mechanism, and excretion of CM-chitosan in rats. After the rats were intraperitoneally injected at the dose of 50 mg/kg, the fluorescein isothiocyanate (FITC)-labeled CM-chitosan was absorbed rapidly and distributed to different organs, including liver, spleen, and kidney. The highest level of CM-chitosan was found in liver. It was at the level of 1.6 ± 0.6 mg/liver and made up approximately 10−22% of the total injected FTC-CM-chitosan. Urinary excretion was the predominant way of excretion of FITC-labeled CM-chitosan, and 85% of the dose was excreted in urine over the period of 11 days. The molecular weights of body distributed FTC-CM-chitoasan and urinary excreted FTC-CM-chitoasan were analyzed by gel chromatography. The results indicated that the FTC-CM-chitosan was degraded in abdominal dropsy. The absorbed CM-chitosan forms were found with a relatively high molecular weight (∼300 kDa), whereas the molecular weight of the urinary excreted FTC-CM-chitosan was less than 45 kDa. In vitro research revealed that the CM-Chi was also degradable in plasma and homogenate of liver. The CM-chitosan with a molecular weight of ∼800k was thoroughly degraded to a small molecule after it was incubated in homogenate of liver at 37 °C for 24 h. The results suggested that the liver plays a central role in biodegradation of CM-chitosan. The excellent biodegradability of CM-chitosan could potentially contribute to the clinical applications. The results also provide important clues for further modification of CM-chitosan as the postsurgical and other biomedical materials. [ABSTRACT FROM AUTHOR]

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0–9.0 and AG-b was stable in the pH range of 4.0–10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 °C, respectively. AG-a was stable at temperature below 50 °C. AG-b was stable at temperature below 60 °C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose. [ABSTRACT FROM AUTHOR]