JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.118 cover

 

Journal of Bioscience and Bioengineering – Recent Articles

  • PLE-wu, a new member of piggyBac transposon family from insect, is active in mammalian cells
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Chunxiao Wu , Shu Wang

    piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity.





  • Characterization of the low-temperature activity of Sulfolobus tokodaii glucose-1-dehydrogenase mutants
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Taisuke Sugii , Satoshi Akanuma , Sota Yagi , Kazuki Yagyu , Yukiko Shimoda , Akihiko Yamagishi

    Thermophilic enzymes are potentially useful for industrial processes because they are generally more stable than are mesophilic or psychrophilic enzymes. However, a crucial drawback for their use in such processes is that most thermophilic enzymes are nearly inactive at moderate and low temperatures. We have previously proposed that modulation of the coenzyme-binding pocket of thermophilic dehydrogenases can produce mutated proteins with enhanced low-temperature activities. In the current study, we produced and characterized mutants of an NADP-dependent glucose-1-dehydrogenase from the hyperthermophile Sulfolobus tokodaii in which a predicted coenzyme-binding, non-polar residue was replaced by another non-polar residue. Detailed analyses of the kinetic properties of the wild-type enzyme and its mutants showed that one of the mutants (V254I) had improved k cat and k cat/K m values at both 25°C and 80°C. Temperature-induced unfolding experiments showed that the thermal stability of the mutant enzyme was comparable to that of the wild-type enzyme. Calculation of the energetic contribution of the V254I mutation for the dehydrogenase reaction revealed that the mutation destabilizes the enzyme-NADP+-glucose ternary complex and reduces the transition-state energy, thus enhancing catalysis.





  • Novel thrombolytic protease from edible and medicinal plant Aster yomena (Kitam.) Honda with anticoagulant activity: Purification and partial characterization
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Jun-Hui Choi , Dae-Won Kim , Se-Eun Park , Bong-Suk Choi , Kumar Sapkota , Seung Kim , Sung-Jun Kim

    A thrombolytic protease named kitamase possessing anticoagulant property was purified from edible and medicinal plant Aster yomena (Kitam.) Honda. Kitamase showed a molecular weight of 50 kDa by SDS-PAGE and displayed a strong fibrin zymogram lysis band corresponding to the similar molecular mass. The enzyme was active at high temperatures (50°C). The fibrinolytic activity of kitamase was strongly inhibited by EDTA, EGTA, TPCK and PMSF, inhibited by Zn2+. The Km and V max values for substrate S-2251 were determined as 4.31 mM and 23.81 mM/mg respectively. It dissolved fibrin clot directly and specifically cleaved the α, Aα and γ-γ chains of fibrin and fibrinogen. In addition, kitamase delayed the coagulation time and increased activated partial thromboplastin time and prothrombin time. Kitamase exerted a significant protective effect against collagen and epinephrine induced pulmonary thromboembolism in mice. These results suggest that kitamase may have the property of metallo-protease like enzyme, novel fibrino(geno)lytic enzyme and a potential to be a therapeutic agent for thrombosis.





  • Characterization of α-1,3-glucanase isozyme from Paenibacillus glycanilyticus FH11 in a new subgroup of family 87 α-1,3-glucanase
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Wasana Suyotha , Shigekazu Yano , Takafumi Itoh , Hiroko Fujimoto , Takao Hibi , Takashi Tachiki , Mamoru Wakayama

    Two α-1,3-glucanase isozymes, designated as α-1,3-glucanase 1 (Agl-FH1) and α-1,3-glucanase 2 (Agl-FH2), were purified from the culture medium of Paenibacillus glycanilyticus FH11. Agl-FH1 and Agl-FH2 exhibited similar characteristics such as optimal pH, pH stability, optimal temperature, thermostability, and molecular masses on SDS-PAGE. However, their hydrolysis products of α-1,3-glucan varied somewhat. Agl-FH1 hydrolyzed α-1,3-glucan into a mixture of maltotriose and maltotetraose, and maltotetraose was the major hydrolysis product of Agl-FH2. N-terminal amino acid sequence analysis and LC–MS/MS analysis of trypsin digested fragments revealed several differences between the amino acid sequences of Agl-FH1 and Agl-FH2. Genes of Agl-FH1 and Agl-FH2 were subcloned into an expression plasmid, and both enzymes were successfully expressed in Escherichia coli. The recombinant Agl-FH1 and Agl-FH2 exhibited the same enzymatic properties as those of each wild-type enzyme, and both of the recombinants showed the activity on the protoplast formation of Schizophyllum commune mycelia. A great diversity was detected in the C-terminal region of family 87 α-1,3-glucanases. Compared with Agl-FH2 which is highly sequence-related to the known α-1,3-glucanases, the C-terminal region of Agl-FH1 has only slight similarity to them (approximately 20% identity). Our analysis revealed that Agl-FH1 was the first member of a new subgroup of family 87 α-1,3-glucanases.





  • Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Nga T. Nguyen , Takeshi Yatabe , Ki-Seok Yoon , Seiji Ogo

    Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the V max of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.





  • Biochemical characterization of Aspergillus oryzae native tannase and the recombinant enzyme expressed in Pichia pastoris
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Toshiyuki Mizuno , Yoshihito Shiono , Takuya Koseki

    In this study, the biochemical properties of the recombinant tannase from Aspegillus oryzae were compared with those of the native enzyme. Extracellular native tannase was purified from a commercial enzyme source. Recombinant tannase highly expressed in Pichia pastoris was prepared as an active extracellular protein. Purified native and recombinant tannases produced smeared bands with apparent molecular masses of 45–80 kDa and 45–75 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, the native enzyme yielded molecular masses of 33 kDa and 30 kDa, whereas the recombinant enzyme yielded molecular masses of 34 kDa and 30 kDa. Purified native and recombinant tannases had an optimum pH of 4.0–5.0 and 5.0, respectively, and were stable up to 40°C. After N-deglycosylation, both enzymes exhibited reduced thermostability. Catalytic efficiencies of both purified enzymes were greater with natural substrates, such as (−)-catechin, (−)-epicatechin, and (−)-epigallocatechin gallates, than those with synthetic substrates, such as methyl, ethyl, and propyl gallates. However, there were no activities against the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids, which indicate feruloyl esterase activity, or the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid, which indicate paraben hydrolase activity.





  • Antioxidant activities and phenolics of fermented Bletilla formosana with eight plant pathogen fungi
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Jianwei Dong , Lixing Zhao , Le Cai , Haixian Fang , Xiuhua Chen , Zhongtao Ding

    The tubers of Bletilla formosana were fermented with eight plant pathogen fungi, respectively, and antioxidant activities and total phenolic content (TPC) of the crude extracts of fermented products and non-fermented products were investigated. The antioxidant activities were evaluated in three different test systems [DPPH, ABTS radical-scavenging activity, and ferric reducing-antioxidant power (FRAP)]. It was found that the extract of Helminthosporium maydis fermented B. formosana (FBF) possessed the highest TPC and exhibited a significant antioxidant activity compared with non-fermented product and other fermented products. Correlation analysis between antioxidant activities and TPC was also investigated. The good correlation between antioxidant activities and TPC revealed that the phenolic compounds might be the major contributors for the high antioxidant activities of the fermented B. formosana. Two phenolic compounds, curvularin and dehydrocurvularin, were isolated from H. maydis FBF, which had never been reported from plant of orchidaceae or H. maydis. Curvularin exhibited significant antioxidant activities, and was also present at a high concentration (0.373 mg/mg extract sample), implying an important role for the antioxidant activity of H. maydis FBF. This study suggested that proper fermentation processing could improve TPC and antioxidant activities of B. formosana.





  • Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Minh Hong Nguyen , Yoshihiro Ojima , Makiko Sakka , Kazuo Sakka , Masahito Taya

    Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.





  • Gas chromatography/mass spectrometry based component profiling and quality prediction for Japanese sake
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Natsuki Mimura , Atsuko Isogai , Kazuhiro Iwashita , Takeshi Bamba , Eiichiro Fukusaki

    Sake is a Japanese traditional alcoholic beverage, which is produced by simultaneous saccharification and alcohol fermentation of polished and steamed rice by Aspergillus oryzae and Saccharomyces cerevisiae. About 300 compounds have been identified in sake, and the contribution of individual components to the sake flavor has been examined at the same time. However, only a few compounds could explain the characteristics alone and most of the attributes still remain unclear. The purpose of this study was to examine the relationship between the component profile and the attributes of sake. Gas chromatography coupled with mass spectrometry (GC/MS)-based non-targeted analysis was employed to obtain the low molecular weight component profile of Japanese sake including both nonvolatile and volatile compounds. Sake attributes and overall quality were assessed by analytical descriptive sensory test and the prediction model of the sensory score from the component profile was constructed by means of orthogonal projections to latent structures (OPLS) regression analysis. Our results showed that 12 sake attributes [ginjo-ka (aroma of premium ginjo sake), grassy/aldehydic odor, sweet aroma/caramel/burnt odor, sulfury odor, sour taste, umami, bitter taste, body, amakara (dryness), aftertaste, pungent/smoothness and appearance] and overall quality were accurately explained by component profiles. In addition, we were able to select statistically significant components according to variable importance on projection (VIP). Our methodology clarified the correlation between sake attribute and 200 low molecular components and presented the importance of each component thus, providing new insights to the flavor study of sake.





  • Chemically pretreating slaughterhouse solid waste to increase the efficiency of anaerobic digestion
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Cyntia R. Flores-Juarez , Adrián Rodríguez-García , Jesús Cárdenas-Mijangos , Leticia Montoya-Herrera , Luis A. Godinez Mora-Tovar , Erika Bustos-Bustos , Francisco Rodríguez-Valadez , Juan Manríquez-Rocha

    The combined effect of temperature and pretreatment of the substrate on the anaerobic treatment of the organic fraction of slaughterhouse solid waste was studied. The goal of the study was to evaluate the effect of pretreating the waste on the efficiency of anaerobic digestion. The effect was analyzed at two temperature ranges (the psychrophilic and the mesophilic ranges), in order to evaluate the effect of temperature on the performance of the anaerobic digestion process for this residue. The experiments were performed in 6 L batch reactors for 30 days. Two temperature ranges were studied: the psychrophilic range (at room temperature, 18°C average) and the mesophilic range (at 37°C). The waste was pretreated with NaOH before the anaerobic treatment. The result of pretreating with NaOH was a 194% increase in the soluble chemical oxygen demand (COD) with a dose of 0.6 g NaOH per g of volatile suspended solids (VSS). In addition, the soluble chemical oxygen demand/total chemical oxygen demand ratio (sCOD/tCOD) increased from 0.31 to 0.7. For the anaerobic treatment, better results were observed in the mesophilic range, achieving 70.7%, 47% and 47.2% removal efficiencies for tCOD, total solids (TS), and volatile solids (VS), respectively.





  • Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Xiaoyu Zhou , Ying Yu , Jianjun Tao , Long Yu

    Lysozyme acts as an important defensive factor in innate immunity due to its well-recognized bacteriolytic activity. Here we describe the production and performance of human lysozyme-like 6 (LYZL6), a novel human c-type lysozyme homolog. A synthetic codon-optimized cDNA encoding the intact amino acid sequence of LYZL6 was cloned and expressed in Pichia pastoris SMD1168. Bioactive LYZL6 was successfully produced as a single major secreted protein with a molecular weight of 15 kDa, and exhibited bacteriolytic activity against Micrococcus lysodeikticus. The expression conditions were optimized, and the highest expression level of LYZL6 occurred when the recombinant strain was induced with 1.5% methanol under pH 4.5 at 24°C for 96 h. When high cell density fermentation of the recombinant P. pastoris was performed using a fed-batch strategy for totally 125 h in a 30 L fermenter, the dry cell weight and the extracellular lysozyme activity were increased to 116.3 g/L and 2340 U/mL, respectively. The LYZL6 protein concentration was 331 mg/L of fermentation supernatant, and the specific activity of LYZL6 towards M. lysodeikticus was 7069 U/mg. Therefore, we proved that LYZL6 is an antibacterial protein, suggesting a potential application of LYZL6 as an antimicrobial agent, and Pichia expression system for LYZL6 was successful and industrially promising.





  • Comparison of sulfate-reducing and conventional Anammox upflow anaerobic sludge blanket reactors
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Ergo Rikmann , Ivar Zekker , Martin Tomingas , Priit Vabamäe , Kristel Kroon , Alar Saluste , Taavo Tenno , Anne Menert , Liis Loorits , Sergio S.C. dC Rubin , Toomas Tenno

    Autotrophic NH 4 + removal has been extensively researched, but few studies have investigated alternative electron acceptors (for example, SO 4 2 ) in NH 4 + oxidation. In this study, sulfate-reducing anaerobic ammonium oxidation (SRAO) and conventional Anammox were started up in upflow anaerobic sludge blanket reactors (UASBRs) at 36 (±0.5)°C and 20 (±0.5)°C respectively, using reject water as a source of NH 4 + . SO 4 2 or NO 2 , respectively, were applied as electron acceptors. It was assumed that higher temperature could promote the SRAO, partly compensating its thermodynamic disadvantage comparing with the conventional Anammox to achieve comparable total nitrogen (TN) removal rate. Average volumetric NH 4 + N removal rate in the sulfate-reducing UASBR1 was however 5–6 times less (0.03 kg-N/(m3 day)) than in the UASBR2 performing conventional nitrite-dependent autotrophic nitrogen removal (0.17 kg-N/(m3 day)). However, the stoichiometric ratio of NH 4 + removal in UASBR1 was significantly higher than could be expected from the extent of SO 4 2 reduction, possibly due to interactions between the N- and S-compounds and organic matter of the reject water. Injections of N2H4 and NH2OH accelerated the SRAO. Similar effect was observed in batch tests with anthraquinone-2,6-disulfonate (AQDS). For detection of key microorganisms PCR-DGGE was used. From both UASBRs, uncultured bacterium clone ATB-KS-1929 belonging to the order Verrucomicrobiales, Anammox bacteria (uncultured Planctomycete clone Pla_PO55-9) and aerobic ammonium-oxidizing bacteria (uncultured sludge bacterium clone ASB08 “Nitrosomonas”) were detected. Nevertheless the SRAO process was shown to be less effective for the treatment of reject water, compared to the conventional Anammox.





  • Efficient transgene expression by alleviation of translational repression in plant cells
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Kiyotaka Ueda , Renya Okawara , Shotaro Yamasaki , Yuji Sanada , Eri Kinoshita , Arata Yoneda , Taku Demura , Ko Kato

    Global translational repression under abiotic stress influences translation of both endogenous and transgene mRNAs. Even in plant cell culture, hypoxia and nutrient deficient stress arise during the growth process. In this study, we first demonstrated the existence of global translational repression in Arabidopsis T87 cultured cells over a time course following inoculation. Next, we performed genome-wide analysis, which revealed that the translational states of endogenous mRNAs differed significantly between growth and stationary phase cells. This analysis showed that translation from most mRNAs was repressed upon stationary phase. Otherwise, a part of mRNA including alcohol dehydrogenase (ADH) gene was recalcitrant to the repression. Furthermore, by polysome analysis and followed quantitative reverse transcription PCR analysis of transformants having 5′untranslated regions (UTRs) of ADH or translationally repressed At3g47610 mRNA fused to reporter gene, we demonstrated that polysomal associations of reporter mRNAs were in accordance with those the mRNAs from which their 5′UTR derived, suggesting that the 5′UTR is an important determinant of the translational state of mRNAs in stationary phase cells. Finally, we demonstrated the effectiveness of 5′UTR of ADH mRNA in transformants derived from the BY-2 tobacco cell line. These results suggested that 5′UTR of ADH mRNA would be a useful element for efficient transgene expression upon stationary phase.





  • Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Nobuyuki Maruyama , Keigo Fujiwara , Kazunori Yokoyama , Cerrone Cabanos , Hisakazu Hasegawa , Kyoko Takagi , Keito Nishizawa , Yuriko Uki , Takeshi Kawarabayashi , Mikio Shouji , Masao Ishimoto , Teruhiko Terakawa

    There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.





  • Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-d-mannose 4,6-dehydratase and β-1,2-xylosyltransferase genes
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Kouki Matsuo , Uiko Kagaya , Noriko Itchoda , Noriko Tabayashi , Takeshi Matsumura

    Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Lea) epitope, Galβ(1–3)[Fucα(1–4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Lea epitopes by repressing the GDP-d-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-l-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264–281, 2011). In the present study, we generated a core β-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of β-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.





  • Micropatterned culture of HepG2 spheroids using microwell chip with honeycomb-patterned polymer film
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Hidekazu Yamazaki , Shun Gotou , Koju Ito , Souichi Kohashi , Yuki Goto , Yukiko Yoshiura , Yusuke Sakai , Hiroshi Yabu , Masatsugu Shimomura , Kohji Nakazawa

    Microwell chip culture is a promising technique for the generation of homogenous spheroids. We investigated the relationship between the structure of the bottom surface of microwell chip and the properties of HepG2 spheroid. We developed a microwell chip, the bottom surface of which consisted of a honeycomb-patterned polymer film (honeycomb film) that had a regular porous structure (HF chip). The chip comprised 270 circular microwells; each microwell was 600 μm in diameter and 600 μm in depth. At the center of the honeycomb film, an area, 200 μm in diameter, was modified with collagen to facilitate cell adhesion. With the exception of the collagen-coated area, the entire microwell was modified with polyethylene glycol to eliminate cell adhesion. HepG2 cells formed uniform spheroids when cultured in the microwells of HF chip. Furthermore, the cells passed through the porous structure of honeycomb film and formed spheroids at its opposite side. The spheroid growth of HepG2 cells cultured in HF chip was greater than that when the cells were culture in a microwell chip, the bottom surface of which was made of poly-methylmethacrylate (PMMA chip). The albumin secretion activity of HepG2 spheroids in HF chip was equal to that in PMMA chip. These results indicate that the microwell bottom with a porous structure enhances the cell growth and maintains well the spheroid function. Thus, HF chip is a promising platform for spheroid cell culture.





  • Differential regeneration of myocardial infarction depending on the progression of disease and the composition of biomimetic hydrogel
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): So Jeong Yoon , Soyoung Hong , Yong Hu Fang , Myeongjin Song , Kuk Hui Son , Ho Sung Son , Sook Kyoung Kim , Kyung Sun , Yongdoo Park

    Hydrogel has been used for regenerating myocardial infraction (MI) as a delivery vehicle for cells and growth factors. This study showed that injectable hyaluronic acid (HA)-based hydrogels alone would effectively regenerate the damaged infarcted heart tissue. We found that there are two major factors of regeneration in MI. One is molecular weight of HA and another is the progression of MI; sub-acute and chronic. Rat MI model was prepared by ligating the left anterior descending coronary artery (LAD). Four weeks after injection of hydrogel, functional analysis of the heart and histological analysis was assessed. When different molecular weight HA-based hydrogels with 50 kDa, 130 kDa, and 170 kDa were applied to the infarcted area in the sub-acute model, 50 kDa HA-based hydrogel showed the most significant regeneration of myocardium as well as functional recovery among samples. For the disease progression, 50 kDa HA-based hydrogels were injected to sub-acute and chronic MI models. The regeneration activity was significantly decreased in the chronic models reflecting that injection timing of the therapeutic agents is also major determinants in the regeneration process. These results suggest that injection time and composition of hydrogel are two major points treating MI.





  • Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Beom-Su Kim , Hark-Mo Sung , Hyung-Keun You , Jun Lee

    Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering.





  • Dietary trimethylamine N-oxide exacerbates impaired glucose tolerance in mice fed a high fat diet
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Xiang Gao , Xiaofang Liu , Jie Xu , Changhu Xue , Yong Xue , Yuming Wang

    Trimethylamine N-oxide (TMAO) is an oxidation product of trimethylamine (TMA) and is present in many aquatic foods. Here, we investigated the effects of TMAO on glucose tolerance in high fat diet (HFD)-fed mice. Male C57BL/6 mice were randomly assigned to the control, high fat (HF), and TMAO groups. The HF group was fed a diet containing 25% fat, and the TMAO group was fed the HFD plus 0.2% TMAO for 4 weeks. After 3 weeks of feeding, oral glucose tolerance tests were performed. Dietary TMAO increased fasting insulin levels and homeostasis model assessment-estimated insulin resistance (HOMA-IR) and exacerbated the impaired glucose tolerance in HFD-fed mice. These effects were associated with the expression of genes related to the insulin signal pathway, glycogen synthesis, gluconeogenesis and glucose transport in liver. mRNA levels of the pro-inflammatory cytokine MCP-1 increased significantly and of the anti-inflammatory cytokine IL-10 greatly decreased in adipose tissue. Our results suggest that dietary TMAO exacerbates impaired glucose tolerance, obstructs the hepatic insulin signaling pathway, and causes adipose tissue inflammation in mice fed a high fat diet.





  • Autonomous bottom-up fabrication of three-dimensional nano/microcellulose honeycomb structures, directed by bacterial nanobuilder
    Publication date: October 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 4

    Author(s): Tetsuo Kondo , Wakako Kasai

    We investigated the autonomous bottom-up fabrication of three-dimensional honeycomb cellulose structures, using Gluconacetobacter xylinus as a bacterial nanoengine, on cellulose honeycomb templates prepared by casting water-in-oil emulsions on glass substrates (Kasai and Kondo, Macromol. Biosci., 4, 17–21, 2004). The template film had a unique molecular orientation state along the honeycomb frames, but was non-crystalline. When G. xylinus, used as a nanofiber-producing bacterium, was incubated on the honeycomb scaffold in a culture medium, it secreted cellulose nanofibers only on the upper surface of the honeycomb frame. The movement was regulated by a selective interaction between the synthesized nanofiber and the surface of the honeycomb frames of the template. The relationship between directed deposition of synthesized nanofibers and ordered fabrication from the nano- to the micro-scale could provide a novel bottom-up methodology, using bacteria, for the design of three-dimensional honeycomb structures as functional materials with nano/micro hierarchical structures, with low energy consumption.