Development of Nanoscale Oil Bodies for Targeted Treatment of Lung Cancer.

Lung cancer is the most widespread disease and is frequently associated with a high level of epidermal growth factor receptor (EGFR). This study was thus conducted to provide a proof-of-concept approach for targeted therapy of lung cancer by development of nanoscale oil bodies (NOBs). This was carried out by fusion of anti-EGFR affibody (ZEGFR2) with oleosin (Ole), a structure protein of plant seed oils. The fusion protein (Ole-ZEGFR2) was produced in Escherichia coli. NOBs were spontaneously assembled from plant oil, phospholipids, and Ole-ZEGFR2. Consequently, Ole-ZEGFR2-based NOBs were selectively internalized by EGFR-positive lung cancer cells with an efficiency exceeding 90%. Furthermore, the hydrophobic anticancer drug, camptothecin (CPT), was encapsulated into Ole-ZEGFR2-based NOBs. The administration of the CPT formulation based on NOBs resulted in a strong antitumor activity both in vitro and in vivo.

Development of Alginate Microspheres Containing Chuanxiong for Oral Administration to Adult Zebrafish.

Oral administration of Traditional Chinese Medicine (TCM) by patients is the common way to treat health problems. Zebrafish emerges as an excellent animal model for the pharmacology investigation. However, the oral delivery system of TCM in zebrafish has not been established so far. This issue was addressed by development of alginate microparticles for oral delivery of chuanxiong, a TCM that displays antifibrotic and antiproliferative effects on hepatocytes. The delivery microparticles were prepared from gelification of alginate containing various levels of chuanxiong. The chuanxiong-encapsulated alginate microparticles were characterized for their solubility, structure, encapsulation efficiency, the cargo release profile, and digestion in gastrointestinal tract of zebrafish. Encapsulation of chuanxiong resulted in more compact structure and the smaller size of microparticles. The release rate of chuanxiong increased for alginate microparticles carrying more chuanxiong in simulated intestinal fluid. This remarkable feature ensures the controlled release of encapsulated cargos in the gastrointestinal tract of zebrafish. Moreover, chuanxiong-loaded alginate microparticles were moved to the end of gastrointestinal tract after oral administration for 6 hr and excreted from the body after 16 hr. Therefore, our developed method for oral administration of TCM in zebrafish is useful for easy and rapid evaluation of the drug effect on disease.

Effects of recombinant lycopene dietary supplement on the egg quality and blood characteristics of laying quails.

This study was conducted to determine the effect of dietary supplement of bacterial lycopene (BL) produced by Escherichia coli on the egg quality and blood characteristics of laying quails. The antioxidant activity measurement showed that BL exhibited 100% 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity at a concentration of 4.65 µg/ml, which was more effective than butylated hydroxytoluene (BHT) and commercial lycopene (CL). Moreover, seven dietary groups of laying quails consisting of 10 100-day-old quails (Coturnix coturnix japonica) each were fed with the basal diet supplemented with BL, CL or canthaxanthin (CA) for 4 weeks. Consequently, the triglyceride content of yolk was significantly lower in the group with BL and CL supplement. The serum malondialdehyde (MDA) level of the BL- and CA-supplemented groups at 18 mg/kg was lower than the control group. In conclusion, BL has a high antioxidant activity and is promising as a feed additive in the diet of laying quails.

Potential production platform of n-butanol in Escherichia coli.

We proposed a potential production platform of n-butanol in Escherichia coli. First, a butyrate-conversion strain was developed by removal of undesired genes and recruiting endogenous atoDA and Clostridium adhE2. Consequently, this E. coli strain grown on the M9 mineral salt with yeast extract (M9Y) was shown to produce 6.2g/L n-butanol from supplemented butyrate at 36h. The molar conversion yield of n-butanol on butyrate reaches 92%. Moreover, the production platform was advanced by additional inclusion of a butyrate-producing strain. This strain was equipped with a pathway comprising atoDA and heterologous genes for the synthesis of butyrate. Without butyrate, the butyrate-conversion and the butyrate-producing strains were co-cultured in M9Y medium and produced 5.5g/L n-butanol from glucose at 24h. The production yield on glucose accounts for 69% of the theoretical yield. Overall, it indicates a promise of the developed platform for n-butanol production in E. coli.

A useful method integrating production and immobilization of recombinant cellulase.

The development of cellulase-based bioprocess is afflicted by the processing efficiency of enzymes. To address this issue, a method based on artificial oil bodies (AOBs) was proposed to integrate production and immobilization of recombinant cellulase. First, the heterologous endoglucanase (celA), cellobiohydrolase (celK), and ß-glucosidase (gls) genes were individually fused with oleosin, a structural protein of plant seed oils. After expression in Escherichia coli, each fusion protein of insolubility was mixed together with plant oils. AOBs were assembled by subjecting the mixture to sonication. Consequently, active CelA, CelK, and Gls were resumed and co-immobilized on AOBs surface. Finally, the assembly condition (including the protein ratio) and the reaction condition were further optimized by response surface methodology. The resulting AOBs-bound cellulase remained stable for 4 cycles of cellulose-hydrolyzed reactions. Overall, the result shows a promise of this proposed approach for processing recombinant cellulase, which may provide a facile method to investigate optimum combination of cellulase components towards various cellulosic materials.

Reduction of Airway Hyperresponsiveness by KWLL in Dermatophagoides-pteronyssinus-Challenged Mice.

Urine therapy has been commonly practiced in ancient civilizations including those of India, China, and Greece. The traditional Chinese medicine KWLL, the precipitation of human urine, has been used in China to alleviate the symptoms of asthma for thousands of years. However, the mechanism of action by which KWLL exerts its immunotherapy is unclear. This study attempted to elucidate the pharmacology of KWLL in mice that had been challenged recurrently by Dermatophagoides pteronyssinus (Der p). BALB/c mice were orally administered KWLL (1 g/kg) before an intratracheal (i.t.) challenge of Der p. Allergic airway inflammation and remodeling were provoked by repetitive Der p (50 µ g/mice) challenges six times at 1 wk intervals. Airway hypersensitivity, histological lung characteristics, and the expression profiles of cytokines and various genes were assessed. KWLL reduced Der p-induced airway hyperresponsiveness and inhibited eosinophil infiltration by downregulating the protein expression of IL-5 in bronchoalveolar lavage fluid (BALF). It also inhibited neutrophil recruitment by downregulating IL-17A in BALF. KWLL effectively diminished inflammatory cells, goblet cell hyperplasia, and mRNA expression of IL-6 and IL-17A in the lung. The reduction by KWLL of airway inflammatory and hyperresponsiveness in allergic asthmatic mice was mediated via immunomodulation of IL-5, IL-6, and IL-17A.

The Immunomodulatory Effect of You-Gui-Wan on Dermatogoides-pteronyssinus-Induced Asthma.

The traditional Chinese medicine You-Gui-Wan (YGW) contains ten species of medicinal plants and has been used to improve health in remissive states of asthma for hundreds of years in Asia. However, little is known about the immunomodulatory mechanisms in vivo. Therefore, this study investigated the pathologic and immunologic responses to YGW in mice that had been repeatedly exposed to Dermatogoides-pteronyssinus (Der p). YGW reduced Der-p-induced airway hyperresponsiveness and total IgE in serum. It also inhibited eosinophil infiltration by downregulating the protein expression of IL-5 in serum and changed the Th2-bios in BALF by upregulating IL-12. Results of the collagen assay and histopathologic examination showed that YGW reduced airway remodeling in the lung. In addition, after YGW treatment there was a relative decrease in mRNA expression of TGF-ß1, IL-13, eotaxin, RANTES, and MCP-1 in lung in the YGW group. The results of EMSA and immunohistochemistry revealed that YGW inhibited NF-κB expression in epithelial lung cells. YGW exerts its regulative effects in chronic allergic asthmatic mice via its anti-inflammatory activity and by inhibiting the progression of airway remodeling.

Selective delivery of cargo entities to tumor cells by nanoscale artificial oil bodies.

Artificial oil bodies (AOBs) are oil droplets that result from self-assembly of a mixture containing triacylglycerols, phospholipids, and membrane proteins of plant seeds. Owing to their small size, stability, hydrophobic core, biocompatibility, and biodegradability, AOBs were explored to examine their feasibility as a drug delivery carrier. This was approached by fusion sesame oleosin (Ole), the primary membrane protein of seed oil bodies, with a small domain consisting of the arginine-glycine-aspartic acid (RGD) motif. The resulting Ole-RGD fusion protein was overproduced in Escherichia coli and then isolated for reconstitution of AOBs. At the optimal condition, the size of stable AOBs was within the range of 100-400 nm. Furthermore, AOBs entrapped with a hydrophobic fluorescence dye were incubated with human tumor cells. As visualized by fluorescent microscopy and confocal microscopy, the RGD-tagged AOBs were able to selectively target cells expressing the αvß3 integrin. Moreover, these AOBs were effectively internalized and the fluorescence dye that they carried was subsequently released inside the cells. The percentage of cells internalized by AOBs could reach 80% as analyzed by flow cytometry. Taken together, it illustrates a great promise of this proposed approach for targeted delivery of cargo entities to tumor cells.

Medium optimization for the production of recombinant nattokinase by Bacillus subtilis using response surface methodology.

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007, 23, 808-813). With this developed B. subtilis strain, the design of an optimum but cost-effective medium for high-level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett-Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box-Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake-flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 degrees C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost-effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high-level production of recombinant nattokinase in a fermenter.

One-step purification of insoluble hydantoinase overproduced in Escherichia coli.

Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.

Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.