Chitosan nanoparticles attenuate hydrogen peroxide-induced stress injury in mouse macrophage RAW264.7 cells.

This study was carried out to investigate the protective effects of chitosan nanoparticles (CNP) against hydrogen peroxide (H2O2)-induced oxidative damage in murine macrophages RAW264.7 cells. After 24 h pre-incubation with CNP (25-200 µg/mL) and chitosan (CS) (50-200 µg/mL, as controls), the viability loss in RAW264.7 cells induced by H2O2 (500 µM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay (P < 0.05) and decreased in cellular LDH release (P < 0.05). Moreover, CNP also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA) (P < 0.05), restoring activities of endogenous antioxidant including superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (P < 0.05), along with increasing total antioxidant capacity (T-AOC) (P < 0.05). In addition, pre-incubation of CNP with RAW264.7 cells for 24 h resulted in the increase of the gene expression level of endogenous antioxidant enzymes, such as MnSOD and GSH-Px (P < 0.05). At the same concentration, CNP significantly decreased LDH release and MDA (P < 0.05) as well as increased MnSOD, GSH-Px, and T-AOC activities (P < 0.05) as compared to CS. Taken together, our findings suggest that CNP can more effectively protect RAW264.7 cells against oxidative stress by H2O2 as compared to CS, which might be used as a potential natural compound-based antioxidant in the functional food and pharmaceutical industries.

Chitosan nanoparticles act as an adjuvant to promote both Th1 and Th2 immune responses induced by ovalbumin in mice.

The study was conducted to investigate the promoted immune response to ovalbumin in mice by chitosan nanoparticles (CNP) and its toxicity. CNP did not cause any mortality or side effects when mice were administered subcutaneously twice with a dose of 1.5 mg at 7-day intervals. Institute of Cancer Research (ICR) mice were immunized subcutaneously with 25 µg ovalbumin (OVA) alone or with 25 µg OVA dissolved in saline containing Quil A (10 µg), chitosan (CS) (50 µg) or CNP (12.5, 50 or 200 µg) on days 1 and 15. Two weeks after the secondary immunization, serum OVA-specific antibody titers, splenocyte proliferation, natural killer (NK) cell activity, and production and mRNA expression of cytokines from splenocytes were measured. The serum OVA-specific IgG, IgG1, IgG2a, and IgG2b antibody titers and Con A-, LPS-, and OVA-induced splenocyte proliferation were significantly enhanced by CNP (P < 0.05) as compared with OVA and CS groups. CNP also significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines and up-regulated the mRNA expression of IL-2, IFN-γ and IL-10 cytokines in splenocytes from the immunized mice compared with OVA and CS groups. Besides, CNP remarkably increased the killing activities of NK cells activity (P < 0.05). The results suggested that CNP had a strong potential to increase both cellular and humoral immune responses and elicited a balanced Th1/Th2 response, and that CNP may be a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV.

White spot syndrome virus (WSSV) is a highly pathogenic and prevalent virus infecting shrimp and other crustaceans. The potentiality of binary ethylenimine (BEI)-inactivated WSSV against WSSV in crayfish, Procambarus clarkii, was investigated in this study. Efficacy of BEI-inactivated WSSV was tested by vaccination trials followed by challenge of crayfish with WSSV. The crayfish injected with BEI-inactivated WSSV showed a better survival (P<0.05) to WSSV on the 7th and 21st day post-vaccination (dpv) compared to the control. Calculated relative percent survival (RPS) values were 77% and 60% on the 7th and 21st dpv for 2mM BEI-inactivated WSSV, and 63%, 30% on 7th and 21st dpv for 3mM BEI-inactivated WSSV. However, heat-inactivated WSSV did not provide protection from WSSV even on 7th dpv. In the inactivation process WSSV especially their envelope proteins maybe changed as happened to 3mM BEI and heat-inactivated WSSV particles. These results indicate the protective efficacy of BEI-inactivated WSSV lies on the integrity of envelope proteins of WSSV and the possibility of BEI-inactivated WSSV to protect P. clarkii from WSSV.

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice.

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.

Influence of Cu2+-loaded silicate on the growth performance and microflora of crucian carp Carassius auratus.

We investigated the effects of Cu2+-loaded silicate (CLS) on the growth performance, microflora of skin, gill and intestine, and intestinal morphology of crucian carp Carassius auratus. A total of 225 native wild crucian carp, with an average initial body weight of 20 g, were randomly divided into 5 treatment groups using 3 replicate tanks of 15 fish per group. The dietary treatments were (1) basal diet, (2) basal diet + CuSO4, (3) basal diet + silicate, (4) basal diet + 0.5% CLS and (5) basal diet + 50 mg kg(-1) chlortetracycline (CTC, purity 98.8%). The trial lasted for 60 d. We found that body weight increased slightly while feed conversion ratio decreased in the CLS-treated group compared with the control groups. The total number of aerobic bacteria counted in the intestine of carp fed the diet supplemented with the CLS (i.e. Vibrio sp. and E. coli), was significantly lower (p = 0.05) compared with the control groups and the CTC-treated fish, while lactobacillus counts were significantly higher (p = 0.05). Lactobacilli counts of the intestine increased significantly (p = 0.05). However, the microflora of the skin and gill was not affected by the addition of CuSO4, silicate, CLS or CTC. The height of the villi in the proximal, mid and distal intestine mucosa of the silicate- and CLS-treated groups was found to be longer (p = 0.05) compared with the villi of the control or the CTC-treated fish. Supplementation with CuSO4 had no effect on the microflora and the intestinal morphology (p = 0.05). These results indicate that CLS had an antibacterial activity in vivo, which may help protect the intestinal mucosa from invasion of pathogenic bacteria and their toxins.

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos.

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.

Preparation, characterization and adsorption properties of chitosan nanoparticles for eosin Y as a model anionic dye.

The present study dealt with the adsorption of eosin Y, as a model anionic dye, from aqueous solution using chitosan nanoparticles prepared by the ionic gelation between chitosan and tripolyphosphate. The nanoparticles were characterized by atomic force microscopy (AFM), size and zeta potential analysis. A batch system was applied to study the adsorption of eosin Y from aqueous solution by chitosan nanoparticles. The results showed that the adsorption of eosin Y on chitosan nanoparticles was affected by contact time, eosin Y concentration, pH and temperature. Experimental data followed Langmuir isotherm model and the adsorption capacity was found to be 3.333 g/g. The adsorption process was endothermic in nature with an enthalpy change (DeltaH) of 16.7 kJ/mol at 20-50 degrees C. The optimum pH value for eosin Y removal was found to be 2-6. The dye was desorbed from the chitosan nanoparticles by increasing the pH of the solution.

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes.

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.

Changes of porcine growth hormone and pituitary nitrogen monoxide production as a response to cadmium toxicity.

The present study was designed to investigate the effects of various cadmium concentrations on porcine growth hormone (GH) secretion in serum and cultured pituitary cells and to explore the possible mechanisms of cadmium toxicity. In feeding trial, 192 barrows (Duroc x Landrace x Yorkshire), with similar initial body weights, were randomly divided into four different treatment groups with three replicates for each treatment. The diets were supplemented for 83 days with 0, 0.5, 5.0, and 10.0 mg/kg cadmium (as CdCl2). For the cell culture trial, dispersed pituitary cells were incubated with graded doses of cadmium (0, 5, 10, 15, or 20 microM) for 24 h. Pigs treated with 10 mg/kg cadmium had significantly decreased serum GH content. 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide assay showed that Cd toxicity was dose-dependent. Cell viability was reduced to 50% at 15 microM concentration. Administration of cadmium significantly reduced GH secretion, whereas cellular NO content and inducible nitric oxide synthase activity increased to a certain extent. These findings suggest that the decrease of GH might be related to NO production and to a change of NO signal pathway caused by cadmium.

Effects of chromium nanoparticle dosage on growth, body composition, serum hormones and tissue chromium in Sprague-Dawley rats.

This 6-week study was conducted to evaluate the effects of seven different levels of dietary chromium (Cr) (0, 75, 150, 300, 450, 600, and 1 200 ppb Cr) in the form of Cr nanoparticle (CrNano) on growth, body composition, serum hormones and tissue Cr in Sprague-Dawley (SD) rats. Seventy male SD rats (average initial body weight of (83.2+/-4.4) g) were randomly assigned to seven dietary treatments (n=10). At the end of the trial, body composition was assessed via dual energy X-ray absorptiometry (DEXA). All rats were then sacrificed to collect samples of blood, organs and tissues for determination of serum hormones and tissue Cr contents. The results indicated that lean body mass was significantly increased (P<0.05) due to the addition of 300 and 450 ppb Cr from CrNano. Supplementation of 150, 300, 450, and 600 ppb Cr decreased (P<0.05) percent body fat significantly. Average daily gain was increased (P<0.05) by addition of 75, 150, and 300 ppb Cr and feed efficiency was increased (P<0.05) by supplementation of 75, 300, and 450 ppb Cr. Addition of 300 and 450 ppb Cr decreased (P<0.05) the insulin level in serum greatly. Cr contents in liver and kidney were greatly increased (P<0.05) by the addition of Cr as CrNano in the dosage of from 150 ppb to 1 200 ppb. In addition, Supplementation of 300, 450, and 600 ppb Cr significantly increased (P<0.05) Cr content in the hind leg muscle. These results suggest that supplemental CrNano has beneficial effects on growth performance and body composition, and increases tissue Cr concentration in selected muscles.

The efficacy of recombinant vaccines against white spot syndrome virus in Procambarus clarkii.

The effectiveness of oral, mock-, and immersion vaccination was investigated against white spot syndrome virus (WSSV) in crayfish. The most exposed WSSV envelope proteins VP19 and VP28 were used in different compositions and with different modes of applications. In experiment 1 crayfish were fed recombinant protein coated food pellets for 25 days, in experiment 2 the purified proteins were directly injected to them followed by one booster dose on 5th day and in experiment 3 the crayfish were left immersed in vaccines for 7 h. Experimental crayfish were challenged on 3rd and 21st days after last vaccination. The overall result showed that VP28 group has lowest cumulative mortality percentage accounting 39.6% at 3rd day and 39.83% at 21st day when injected and it was 43.2% and 49% when fed orally and 46.3% and 46.5% when immersed at 3rd and 21st days, respectively (p<0.05). In VP19 and VP28 (50:50) mixture, mock vaccination showed better performance (36.5%) over immersion (53%) and oral vaccination (53.2%) when challenged on 3rd day and mock vaccination (36.50%) followed by oral (51%) and immersion vaccination (56.5%) on 21st day as well. The VP19 recombinant mock vaccination group performed better (52.91% of 3rd day and 56.46% of 21st day) than oral (76% of 3rd day and 82% of 21st day) and immersion (83% of 3rd day and 86.3% of 21st day) vaccine groups. All the experimental groups except VP19 were significantly different (p<0.05) from the control groups. A positive cumulative effect was observed when VP28 was mixed with VP19 in equal proportion in all the experimental trials, which shows the effectiveness of VP19 as a vaccine component too. In the present trial on the basis of cumulative mortality percentage it is found that mock-vaccination group is more effective than the oral vaccination and immersion vaccination. It also suggests that specific memory exists in crayfish and the effects of VP28 are significant. The effect of VP19 along with VP28 has also shown significant effect against WSSV.

Optimization of process parameters for reduction of gossypol levels in cottonseed meal by Candida tropicalis ZD-3 during solid substrate fermentation.

The objective of this work is to optimize the process parameters for detoxification of gossypol in cottonseed meal (CSM) by Candida tropicalis ZD-3 during solid substrate fermentation (SSF). The maximum detoxification efficiency of gossypol was achieved by employing the substrate, which consists of 70% of CSM, 20% of corn flour and 10% of wheat bran. The optimum fermentation conditions for gossypol detoxification are incubation period of 48h, incubation temperature at 30 degrees Celsius, inoculum level 5% v/w, moisture content of solid substrate 50% and pH in nature. Adding minerals solution to CSM substrate benefit fermentation detoxification.

Effects of fluoride on hepatic antioxidant system and transcription of Cu/Zn SOD gene in young pigs.

Thirty-two barrows (Duroc x Landrace x Yorkshire) were randomly divided into four groups, each of which included eight pigs. The groups received the same basal diet supplemented with 0, 100, 250 and 400mg/kg fluoride, respectively. The malondialdehyde (MDA) and glutathione (GSH) levels, antioxidant enzymes activities and zinc/copper superoxide dismutase (Cu/Zn SOD) mRNA content in the liver were determined to evaluate the fluoride hepatic intoxication. Results showed the increased lipid peroxides (LPO) level and the reduced GSH content, along with a concomitant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px). Moreover, the level of hepatic Cu/Zn SOD mRNA was also significantly reduced. We suggest the mechanism of fluoride injuring the liver as follows: fluoride causes a decrease in Cu/Zn SOD mRNA and the reduced activities of antioxidant enzymes, leads to the declined ability of scavenging free radicals with excessive production of LPO, which seriously damages the hepatic structure and function.

Effect of hyperthermia on the replication of white spot syndrome virus (WSSV) in Procambarus clarkii.

The effect of hyperthermia on the development of white spot syndrome virus (WSSV) in the crayfish Procambarus clarkii was studied by competitive PCR. Crayfish were exposed to different temperatures (24 +/- 1 and 32 +/- 1 degrees C) after WSSV injection. No mortality was observed when crayfish were held at 32 +/- 1 degrees C, but mortality reached 100% when crayfish were transferred to 24 +/- 1 degrees C. Competitive PCR showed that viral levels at 32 +/- 1 degrees C remained at 10(5) copies mg(-1) tissue, while at 24 +/- 1 degrees C levels were significantly higher, rising from 10(4) to 10(10) copies mg(-1) tissue. These results suggest that hyperthermia reduces viral replication, but does not eliminate viral particles from WSSV-infected crayfish.

Effect of cadmium on lipid peroxidation and activities of antioxidant enzymes in growing pigs.

Malondialdehyde (MDA), glutathione (GSH) content, total antioxidant capacity (T-AOC) levels, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione transferase (GST) activities were studied in serum, liver, and kidney of growing pigs after graded doses of cadmium administration in diets. One hundred ninety-two barrows (Duroc x Landrace x Yorkshire), with similar initial body weight 27.67 +/- 1.33 kg, were randomly allotted into 4 different treatments with 3 replications (16 pigs per replication). The treatments received the same basal diet added with 0, 0.5, 5.0, and 10.0 mg/kg cadmium (as CdCl2), respectively. The results showed pigs treated with 10 mg/kg cadmium significantly decreased average daily gain (ADG) (p<0.05) and increased feed/gain ratio (F/G) (p<0.05) compared to the control. In this treatment, the contents of MDA increased significantly (p<0.05), GSH concentrations, T-AOC levels, and the activities of SOD, GSH-PX, and GST decreased significantly (p<0.05). The results indicate 10 mg/kg cadmium could decrease pig antioxidant capacity after extended exposure and cadmium-induced increase lipid peroxidation might not be only the result of the possibility of lower level of GSH but could also be as a result of direct action of cadmium on peroxidation reaction.

Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal.

The objective of this work was to investigate the effect of six individual strains of fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal (CSM). Six groups of disinfected CSM substrate were incubated for 48 h after inoculation with either of the fungi C. capsuligena ZD-1, C. tropicalis ZD-3, S. cerevisae ZD-5, A. terricola ZD-6, A. oryzae ZD-7, or A. niger ZD-8. One not inoculated group (substrate) was used as a control. Levels of initial and final free gossypol (FG), crude protein (CP), amino acids (AA) and in vitro digestibility were assayed. The experiment was done in triplicate. The experimental results indicated that microbial fermentation could greatly decrease (P<0.05) FG levels in CSM. The detoxification efficiency differed between the species of microorganisms applied. From the perspective of reducing CSM potential toxicity, C. tropicalis ZD-3 was most successful followed by S. cerevisae ZD-5 and A. niger ZD8. They could reduce FG levels of CSM to 29.8, 63.07 and 81.50 mg/kg based on DM (dry matter), respectively, and their detoxification rate were 94.57%, 88.51% and 85.16%, respectively. If crude protein, amino acids content and their in vitro digestibility were also taken into account, A. niger ZD-8 may be the best choice. The CP content of CSM substrate fermented by C. tropicalis ZD-3 and A. niger ZD-8 were improved by 10.76% and 22.24%; the TAA (total amino acids) contents were increased by 7.06% and 11.46%, and the EAA (essential amino acids) were raised by 7.77% and 12.64%, respectively. Especially, the levels of methionine, lysine and threonine were improved greatly (P<0.05). The in vitro CP digestibility of CSM fermented by C. tropicalis ZD-3 and A. niger ZD-8 was improved by 13.42% and 18.22%, the TAA were increased by 17.75% and 22.88%, and the EAA by 16.61% and 21.01%, respectively. In addition, the in vitro digestibility of methionine, lysine and threonine was also improved greatly (P<0.05).

In vitro effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 cells.

AIM: To investigate the effects of chitosan nanoparticles on proliferation of human gastric carcinoma cell line MGC803 in vitro and the possible mechanisms involved. METHODS: Chitosan nanoparticles were characterized by particle size, zeta potential, and morphology. After treatment with various concentrations of chitosan nanoparticles (25, 50, 75, 100 microg/mL) at various time intervals, cell proliferation, ultrastructural changes, DNA fragmentation, mitochondrial membrane potential (MMP), cell cycle phase distribution and apoptotic peaks of MGC803 cells were analyzed by MTT assay, electron microscopy, DNA agarose gel electrophoresis, and flow cytometry. RESULTS: Chitosan nanoparticles exhibited a small particle size as 65 nm and a high surface charge as 52 mV. Chitosan nanoparticles markedly inhibited cell proliferation of MGC803 cells with an IC50 value of 5.3 microg/mL 48 h after treatment. After treatment with chitosan nanoparticles, the typical necrotic cell morphology was observed by electron microscopy, a typical DNA degradation associated with necrosis was determined by DNA agarose electrophoresis. Flow cytometry showed the loss of MMP and occurrence of apoptosis in chitosan nanoparticles-treated cells. CONCLUSION: Chitosan nanoparticles effectively inhibit the proliferation of human gastric carcinoma cell line MGC803 in vitro through multiple mechanisms, and may be a beneficial agent against human carcinoma.

Production of human single-chain variable fragment (scFv) antibody specific for digoxin by ribosome display.

Ribosome display was applied in vitro to select single-chain variable fragment (scFv) antibody specific for digoxin from a human non-immune naive scFv library. A cell-free system was used to produce stable antibody-ribosome-mRNA (ARM) complexes to provide the linkage of genotype and phenotype, allowing simultaneous selection of a desired antibody together with its encoding mRNA. The mRNA was then recovered and amplified as DNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Repeating the display cycle enriched the selected molecules, enabling rare species to be isolated. In this study, digoxin-binding segments were selected over four cycles of ARM display and the selected DNA was cloned and expressed as a single-chain variable fragment antibody (the best scFv, A3) in Escherichia coli. The affinity (equilibrium dissociation constant Kd) of digoxin was 8.3 x 10(-8) M for A3, which validated construction of the naïve library and the power of ribosome display lending to the evolution of functional characteristics, such as potency of leading candidate antibodies to provide therapeutic antibodies. A3 was purified using affinity chromatography and determined by Western blot. The results indicate that ribosome display technnique can be efficiently used to isolate specific antibody fragments from a naive library.

Production of recombinant enveloped structural proteins from the Chinese WSSV isolate.

The white spot syndrome virus (WSSV) is one of the deadly pathogens of penaeid shrimps and other crustaceans. The WSSV virion consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double stranded DNA genome of 305 Kb with 181 open reading frames. The two major structural genes, VP19 and VP28 were amplified from the genomic DNA of Chinese isolate of WSSV and cloned in pUCm-T vector and sub cloned in pET-30a (+) vector. The expressions of genes inE. coli (BL21) were confirmed by SDS-PAGE analysis. The clones were sequenced, submitted to the gene bank and the Xiang Shan strain of WSSV were compared with the previous reported sequence of WSSV of various regions which revealed that VP19 and VP28 gene sequences had certain differences from the sequences of similar genes of the isolate already reported. The recombinant proteins expressed, purified and characterized.