Astaxanthin Protects against Hyperglycemia-Induced Oxidative and Inflammatory Damage to Bone Marrow and to Bone Marrow-Retained Stem Cells and Restores Normal Hematopoiesis in Streptozotocin-Induced Diabetic Mice.

Hyperglycemia has various adverse health effects, some of which are due to chronic oxidative and inflammatory impairment of bone marrow (BM), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). Astaxanthin (ASTX) has been shown to ameliorate hyperglycemia-associated systemic complications and acute mortality, and this effect is partially associated with restoration of normal hematopoiesis. Here, the effects of ASTX on diabetes-induced complications in BM and BM stem cells were investigated, and the underlying molecular mechanisms were elucidated. Ten-week-old C57BL/6 mice received a single intraperitoneal injection of streptozotocin (STZ; 150 mg/kg) in combination with oral gavage of ASTX (12.5 mg/kg) for 30 or 60 consecutive days. Supplemental ASTX ameliorated acute mortality and restored the STZ-impaired bone mass accrual and BM microenvironment in STZ-injected mice. Oral gavage of ASTX suppressed osteoclast formation in the BM of STZ-injected mice. Specifically, supplementation with ASTX inhibited oxidative stress and senescence induction of BM HSCs and MSCs and ameliorated hematopoietic disorders in STZ-injected mice. These effects of ASTX were associated with BM restoration of angiopoietin 1, stromal cell-derived factor 1, ß-catenin, and Nrf2. Long-term ASTX gavage also recovered the STZ-induced dysfunction in migration, colony formation, and mineralization of BM-derived stromal cells. Further, a direct addition of ASTX exhibited direct and dose-dependent inhibition of osteoclastic activation without cytotoxic effects. Collectively, these results indicate that ASTX protects against diabetes-induced damage in the BM microenvironment in BM, HSCs, and MSCs and restores normal hematopoiesis and bone accrual in STZ-injected mice.

Ethanol extract of Dryopteris crassirhizoma alleviates allergic inflammation via inhibition of Th2 response and mast cell activation in a murine model of allergic rhinitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris crassirhizoma (DC) is used as a traditional herbal remedy to treat various diseases, the tapeworm infection, common cold, and cancer in Korea, Japan, and China. DC also has the antioxidant anti-inflammatory and antibacterial activities. However, the anti-allergic inflammatory effect of DC and some of its mechanisms in allergic rhinitis model are unknown well. AIM OF THIS STUDY: The purpose of this study is to investigate the anti-allergic inflammatory effect of DC on the allergic rhinitis model, mast cell activation and histamine release. MATERIALS AND METHODS: Allergic rhinitis was induced in BALB/c mice by sensitization and challenge with ovalbumin (OVA). Different concentration of DC and dexamethasone was administrated by oral gavage on 1 h before the OVA challenge. Mice of the control group were treated with saline only. Then mice were evaluated for the presence of nasal mucosa inflammation, the production of allergen-specific cytokine response and the histology of nasal mucosa. RESULTS: DC significantly ameliorated the nasal symptoms and the inflammation of nasal mucosa. DC also reduced the infiltration of eosinophils and mast cells in these tissues and the release of histamine in blood. Meanwhile, DC evidently inhibited the overproduction of Th2 cytokines and increased the Th1 and Treg cytokines in nasal lavage fluid by OVA. DC also reduced the levels of OVA-specific IgE, IgG1 and IgG2a in blood. CONCLUSIONS: This study suggests that DC has a significant anti-allergic inflammatory effect in the nasal cavity. DC may have the therapeutic effect of allergic rhinitis.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa).

Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.

Expression and assembly of cholera toxin B subunit (CTB) in transgenic carrot (Daucus carota L.).

We expressed the cholera toxin B subunit (CTB) fused to an endoplasmic reticulum retention signal (SEKDEL) in carrot roots using an Agrobacterium-mediated transformation method. Fourteen independent transgenic lines were regenerated via somatic embryogenesis after 6 months of culture. The sCTB gene was detected in the genomic DNA of transgenic carrot by PCR amplification. Expressions and assembly of sCTB protein into oligomeric structures of pentameric size were observed in transgenic plant extracts by Western blot analysis. The sCTB produced by transgenic carrot roots demonstrated strong affinity for GM1-ganglioside, suggesting that the sCTB conserved the antigenic sites for binding and proper folding of the pentameric sCTB structure. The expression level of sCTB comprised approximately 0.48% of total soluble protein (TSP) in root of transgenic carrot.

Isolation and characterization of antioxidation enzymes from cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 5-l bioreactor.

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40 degrees C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60 degrees C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.

Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa).

Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.

Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato (Solanum tuberosum) cells and the ctb-fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21 kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB-FimA (266-337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB-FimA fusion protein was confirmed by ELISA measurements of GM1 ganglioside receptor binding. Quantification of the CTB-FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB-FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease.

Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato.

A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.

HIV-1 gp120 V3 cholera toxin B subunit fusion gene expression in transgenic potato.

A cDNA fragment encoding the V3 loop of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 was fused to the cholera toxin B subunit gene (CTB-gp120) and transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-gp120 fusion gene was detected in genomic DNA from transformed potato leaves by PCR DNA amplification. Synthesis and assembly of the CTB-gp120 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-gp120 fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that CTB-gp120 fusion protein made up 0.002-0.004% of the total soluble tuber protein. Synthesis of CTB-gp120 monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates for the first time the expression of HIV-1 gp120 in plants and emphasizes the feasibility of using edible plant-based vaccination for protection against HIV-1 infection.

SIVmac Gag p27 capsid protein gene expression in potato.

A cDNA encoding the Simian immunodeficiency virus type (SIV(mac)) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification. Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues. Based on ELISA results, recombinant Gag protein made up 0.006-0.014% of total soluble tuber protein. The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection.

Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato.

A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6mg per 100g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection.