Metabolic engineering of Agrobacterium sp. ATCC31749 for curdlan production from cellobiose.

Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing ß-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation.

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig-human xenotransplantation.

O-GlcNAc protein modification in cancer cells increases in response to glucose deprivation through glycogen degradation.

When cellular glucose concentrations fall below normal levels, in general the extent of protein O-GlcNAc modification (O-GlcNAcylation) decreases. However, recent reports demonstrated increased O-GlcNAcylation by glucose deprivation in HepG2 and Neuro-2a cells. Here, we report increased O-GlcNAcylation in non-small cell lung carcinoma A549 cells and various other cells in response to glucose deprivation. Although the level of O-GlcNAc transferase was unchanged, the enzyme contained less O-GlcNAc, and its activity was increased. Moreover, O-GlcNAcase activity was reduced. The studied cells contain glycogen, and we show that its degradation in response to glucose deprivation provides a source for UDP-GlcNAc required for increased O-GlcNAcylation under this condition. This required active glycogen phosphorylase and resulted in increased glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting enzyme in the hexosamine biosynthetic pathway. Interestingly, glucose deprivation reduced the amount of phosphofructokinase 1, a regulatory glycolytic enzyme, and blocked ATP synthesis. These findings suggest that glycogen is the source for increased O-GlcNAcylation but not for generating ATP in response to glucose deprivation and that this may be useful for cancer cells to survive.

Large and small extracellular vesicles released by glioma cells in vitro and in vivo.

Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative-polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro-nuclear magnetic resonance (µNMR). We report that both U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87EGFRvIII glioma-bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87EGFRvIII cells and from serum of U87EGFRvIII glioma-bearing mice. Both large and small EVs contain tumour-specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.

Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation.

Gangliosides are a family of sialic acid-containing glycosphingolipids that are abundant in neurons and have a variety of functions in developing and mature tissues. We examined the expression of ganglioside GT1b in the embryonic preimplantation stage after freezing and thawing processes to determine the regulatory roles of ganglioside GT1b in early embryonic development. ICR mouse embryos at the two-cell stage obtained by flushing the oviducts were frozen by two cryopreservation procedures, slow freezing using a programmable freezer or vitrification by direct plunging into liquid nitrogen. Slow freezing was conducted with equilibration in 1.5 M 1,2-propanediol or 5% equilibration glycerol. Vitrification was applied with a 10-15 min equilibration in 7.5% ethylene glycol (EG), 7.5% dimethylsulfoxide (DMSO), and 30 sec in a solution of 15% EG, 15% DMSO and 0.5 M sucrose. Immediately after thawing, the survival rate of the embryos was assessed by their morphology and ability to develop to blastocysts in culture. The survival rate of vitrified and thawed embryos (92%) was significantly higher than that of slow frozen and thawed embryos (76%) (P<0.05). A tendency of higher blastocyst rate was found in the vitrified and thawed embryos compared to that of the slow frozen and thawed embryos. Confocal immunofluorescence staining confirmed that surviving embryos expressed ganglioside GT1b, with the strongest expression at the compacted eight-cell or later stage embryos. Ganglioside GT1b was not observed in the TUNEL-positive, apoptotic embryos, suggesting that cryopreservation had induced DNA breaks in them. These results suggest that ganglioside GT1b may play an important role in embryo survival or development.

Isolation and characterization of marine bacterial strain degrading fucoidan from Korean Undaria pinnatifida Sporophylls.

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct lowmolecular- mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer Lfucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an alpha-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.

Increased alpha2,3-sialylation and hyperglycosylation of N-glycans in embryonic rat cortical neurons during camptothecin-induced apoptosis.

Alterations in the glycan chains of cell surface glycoconjugates are frequently involved biological processes such as cell-cell interaction, cell migration, differentiation and development. Cultured embryonic (E18) rat cortical neurons underwent apoptosis in response to camptothecin, and lectin histochemistry showed that binding to apoptotic neurons of FITC-conjugated Maackia amurensis agglutinin (MAA), which is specific for terminal alpha2,3-sialic acid residues, increased progressively with increasing concentrations of camptothecin. Analysis of the total proteins of apoptotic neurons by SDS-PAGE, and lectin blotting using HRP-labeled MAA, revealed that the expression of terminal alpha2,3-sialic acid residues on an unknown protein with an apparent molecular mass of 25.6 kDa also increased in apoptotic neurons. NP-HPLC analysis of the total cellular N-glycans of normal and apoptotic neurons demonstrated that the expression of structurally simpler biantennary types of N-glycans fell by 49% during apoptosis whereas the more branched triantennary types of N-glycans with terminal sialic acid residues increased by up to 59%. These results suggest that increased surface expression of alpha2,3-sialic acid residues and hyperglycosylation of N-glycans is a common feature of cellular responses to changes in cell physiology such as tumorigenesis and apoptosis.

Moxibustion at ST36 alleviates pain in complete Freund's adjuvant-induced arthritic rats.

This study was to investigate the antinociceptive effects of moxibustion in a complete Freund's adjuvant (CFA)-induced arthritic rat model, and the effects of moxibustion on immunohistochemical changes at the spinal cord level. Moxibustion was applied to the ipsilateral (right) Zusanli (ST36) acupoint to the lesion side for 9 days to CFA-induced arthritic rats. The stepping force was measured as a behavioral test, c-Fos immunohistochemistry, NO production and nNOS Western blots were examined to evaluate antinociceptive effects. Moxibustion at ST36 significantly improved the stepping force in the affected hind limb in CFA-induced arthritis. Moreover, moxibustion at ST36 suppressed the production of NO and the protein expression of c-Fos and nNOS induced by arthritis. These results suggest that moxibustion at ST36 has a potent antinociceptive effect in an arthritic rat model, and modulates neuronal excitability and endogenous NO production by suppressing c-Fos and nNOS protein expression.

Pathophysiological implication of ganglioside GM3 in early mouse embryonic development through apoptosis.

Apoptosis may occur in early embryos where the execution of essential developmental events has failed, and gangliosides, sialic acid-conjugated glycosphingolipids, are proposed to regulate cell differentiation and growth. To evaluate the regulatory roles of ganglioside GM3 in early embryonic development, this study examined its expressional patterns in apoptotic cells during early embryonic development in mice. Pre-implanted embryos were obtained by in vitro fertilization, which were treated at the 4-cell stage with three the apoptosis inducers, actinomycin D, camptothecin and cycloheximide, for 15 h. All three inducers significantly increased the percentage of apoptotic cells, as measured using a TUNEL method, but remarkably reduced the total cell numbers. The numbers of morula and blastocyst stages were significantly decreased by treatment of the embryos with the three apoptosis inducers compared with the control, with a similar result also observed in the number of blastomeres. Staining of early embryos with Hoechst 33342 revealed a significant percentage of apoptotic nuclei. Prominent immunofluorescence microscopy revealed a significant difference in the ganglioside GM3 expression in apoptotic embryos compared with the control, and RT-PCR also demonstrated a dramatic increase in ganglioside GM3 synthase mRNA in the apoptotic embryos. These results suggest that ganglioside GM3 may be pathophysiologically implicated in the regulation of early embryonic development through an apoptotic mechanism.

Biodegradation-tunable mesoporous silica nanorods for controlled drug delivery.

Mesoporous silica in the forms of micro- or nanoparticles showed great potentials in the field of controlled drug delivery. However, for precision control of drug release from mesoporous silica-based delivery systems, it is critical to control the rate of biodegradation. Thus, in this study, we demonstrate a simple and robust method to fabricate "biodegradation-tunable" mesoporous silica nanorods based on capillary wetting of anodic aluminum oxide (AAO) template with an aqueous alkoxide precursor solution. The porosity and nanostructure of silica nanorods were conveniently controlled by adjusting the water/alkoxide molar ratio of precursor solutions, heat-treatment temperature, and Na addition. The porosity and biodegradation kinetics of the fabricated mesoporous nanorods were analyzed using N2 adsorption/desorption isotherm, TGA, DTA, and XRD. Finally, the performance of the mesoporous silica nanorods as drug delivery carrier was demonstrated with initial burst and subsequent "zero-order" release of anti-cancer drug, doxorubicin.

Levan fructotransferase from Arthrobacter oxydans J17-21 catalyzes the formation of the di-D-fructose dianhydride IV from levan.

A new levan fructotransferase (LFTase) isolated from Arthrobacter oxydans J17-21 was characterized for the production of difructose dianhydride IV (DFA IV). LFTase was purified to apparent homogeneity by Q-Sepharose ion exchange chromatography, Mono-Q HR 5/5 column chromatography, and gel permeation chromatography. The enzyme had an apparent molecular mass of 54000 Da. The optimum pH for the enzyme-catalyzed reaction was pH 6.5, and the optimum temperature was observed at 45 degrees C. The LFTase was activated by the presence of CaCl(2) and EDTA-2Na but inhibited strongly by MnCl(2) and CuSO(4) at 1 mM and completely by FeSO(4) and Ag(2)SO(4) at 1 mM. A bacterial levan from Zymomonas mobilis was incubated with an LFTase; final conversion yield from the levan to DFA IV was 35%. Neither inulin, levanbiose, sucrose, dextran, nor starch was hydrolyzed by LFTase. DFA IV was very stable at acidic pH and high temperature, thus indicating that DFA IV may be suitable for the food industry and related areas.

Protective Effect of Coriolus versicolor Cultivated in Citrus Extract Against Nitric Oxide-Induced Apoptosis in Human Neuroblastoma SK-N-MC Cells.

Nitric oxide (NO) is a reactive free radical and a messenger molecule in many physiological functions. However, excessive NO is believed to be a mediator of neurotoxicity. The medicinal plant Coriolus versicolor is known to possess anti-tumor and immune-potentiating activities. In this study, we investigated whether Coriolus versicolor possesses a protective effect against NO donor sodium nitroprusside (SNP)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. We utilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and caspase-3 enzyme activity assay in SK-N-MC cells. MTT assay showed that SNP treatment significantly reduces the viability of cells, and the viabilities of cells pre-treated with the aqueous extract of Coriolus versicolor cultivated in citrus extract (CVE(citrus)) was increased. However, aqueous extract of Coriolus versicolor cultivated in synthetic medium (CVE(synthetic)) showed no protective effect and aqueous citrus extract (CE) had a little protective effect. The cell treated with SNP exhibited several apoptotic features, while those pre-treated for 1 h with CVE(citrus) prior to SNP expose showed reduced apoptotic features. The cells pre-treated for 1 h with CVE(citrus) prior to SNP expose inhibited p53 and Bax expressions and caspase-3 enzyme activity up-regulated by SNP. We showed that CVE(citrus) exerts a protective effect against SNP-induced apoptosis in SK-N-MC cells. Our study suggests that CVE(citrus) has therapeutic value in the treatment of a variety of NO-induced brain diseases.

Estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized rats.

This study examined the estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized (OVX) rats. The components of silkworm pupae were extracted in distilled water at room temperature for 6 hours. The ovaries of six-week old female rats were then bilaterally removed. One week after OVX, the animals were treated with 200, 400 or 600 mg/kg/day of silkworm pupae extracts. The body weights of the OVX rats increased remarkably compared to the control rats, however their relative uterus weights to body weights decreased significantly. Treatment with the aqueous extracts of silkworm pupae dramatically improved the decreased uterus weights of OVX rats, with the highest increase observed in treatment with 200 mg/kg/day of the aqueous extracts. Additionally, treatment with aqueous extracts (200 mg/kg/day) of silkworm pupae significantly elevated the serum 17beta-estradiol contents of OVX rats when compared to the control animals. To examine the toxic effects of silkworm pupae on the hepatic functions of OVX rats, the levels of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were measured. The serum GOT and GPT levels did not change in response to the administration of aqueous extracts (200, 400 and 600 mg/kg/day) for 4-weeks. Taken together, these results suggest that the aqueous extracts of silkworm pupae may have estrogenic activity, which suggests that silkworm pupae may be useful in the prevention and/or treatment of menopausal disorders caused by deficiencies in female sexual hormones, including estrogen.

Control of cytokine gene expression by PG101, a water-soluble extract prepared from Lentinus lepideus.

A water-soluble extract from Lentinus lepideus mycelium, named PG101, has been shown to control the expression of various cytokines [M. Jin, H.J. Jung, J.J. Choi, H.Jeon, J.H. Oh, B. Kim, S.S. Shin, J.K. Lee, K. Yoon, S. Kim, Activation of selective transcription factors and cytokines by water-soluble extract from Lentinus lepideus, Exp. Biol. Med. (Maywood) 228 (2003) 749-758]. To understand its molecular mechanism(s), PG101-mediated activation of cytokines was studied at the RNA and protein levels. Results from Northern blot analysis indicated that the steady-state RNA levels of TNF-alpha and seven other cytokines were highly increased in human peripheral blood mononuclear cells treated with PG101. The RNA level of TNF-alpha, MIP-1alpha, MIP-1beta, MIP-3alpha, and IL-8 was not affected by the presence of cycloheximide, an inhibitor of the translation process, suggesting that they are the direct targets of PG101. A significantly high protein level of TNF-alpha, MIP-1alpha, and IL-8 remained detectable, even when cells were cultured with actinomycin D, 2h prior to the PG101 treatment. Our data indicate that PG101 controls selective cellular proteins, which play key roles in the innate immune system, at the transcriptional and post-translational levels.

Bioconversion of compactin into pravastatin by Streptomyces sp.

Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l(-1) from 750 mg compactin l(-1) in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l(-1) and 1000 mg pravastatin l(-1), respectively, with the conversion rate of 10 mg l(-1) h(-1). Continuous feeding of compactin increased production of pravastatin to 15 mg l(-1) h(-1).