JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.119 cover

Journal of Bioscience and Bioengineering – Recent Articles

  • Substrate specificity of β-glucosidase from Gordonia terrae for ginsenosides and its application in the production of ginsenosides Rg3, Rg2, and Rh1 from ginseng root extract
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Kyung-Chul Shin , Hye-Ji Lee , Deok-Kun Oh

    A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae β-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.





  • Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Wenjuan Han , Mika Yamauchi , Urara Hasegawa , Masanori Noda , Kiichi Fukui , André J. van der Vlies , Susumu Uchiyama , Hiroshi Uyama

    Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column.





  • Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Yoshio Kimura , Maho Urata , Reiko Okamoto

    Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting.





  • Cloning and heterologous expression of the ftfCNC-2(1) gene from Weissella confusa MBFCNC-2(1) as an extracellular active fructansucrase in Bacillus subtilis
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Amarila Malik , Maria Tyas Hapsari , Iwao Ohtsu , Shu Ishikawa , Hiroshi Takagi

    Fructan-exopolysaccharides (fructan-EPS) (inulin and levan) and their oligosaccharides (fructooligosaccharides, FOS) have drawn considerable interest in the food and pharmaceutical industries. EPS-producing lactic acid bacteria have been reported to produce β-fructans (inulin and levan), as well as α-glucans, by the function of sucrase enzymes, i.e., fructansucrase and glucansucrase. A fructansucrase ftfCNC-2(1) gene from Weissella confusa strain MBFCNC-2(1) was previously cloned in Escherichia coli. In this study, we aimed to express the ftf[CNC-2(1)] gene in Bacillus subtilis to obtain the active form of the extracellular recombinant protein FTF[CNC-2(1)]. This cloning was achieved by inserting the gene in-fusion with the signal sequence of the B. subtilis subtilisin E. SDS-polyacrylamide gel electrophoresis analysis and in situ activity assay with Periodic Acid-Schiff staining revealed that the recombinant FTF[CNC-2(1)] was successfully expressed as an extracellular protein from B. subtilis DB403 in its active form, which was confirmed using sucrose and raffinose.





  • Lactobacillus acidophilus CP23 with weak immunomodulatory activity lacks anchoring structure for surface layer protein
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Sae Yanagihara , Shinji Kato , Nobuhisa Ashida , Naoyuki Yamamoto

    To determine the reason for the low levels of Surface layer protein A (SlpA) on CP23 cells, which might play a crucial role in the immunomodulatory effect of Lactobacillus acidophilus, the DNA sequence of the slpA gene of CP23 and L-92 strains, including the upstream region, were analyzed. Unexpectedly, there was no significant difference in the predicted amino acid sequence of the C-terminus needed for cell anchoring, and only an additional Ala-Val-Ala sequence inserted in the N-terminal region of the mature CP23 protein. Therefore, anchoring of SlpA on the cell wall of CP23 and L-92 was evaluated by a reconstitution assay, which showed that SlpA released by LiCl treatment from both CP23 and L-92 was successfully anchored on LiCl-treated L-92 cells, but not on LiCl-treated CP23 cells. Moreover, quantitative analysis of SlpA protein in the culture medium of CP23 and L-92 by ELISA revealed higher levels of SlpA secretion in CP23 cells than in L-92 cells. Collectively, these results suggest that the lower levels of SlpA on the surface of CP23 cells might be caused by less cell wall capacity for SlpA anchoring, leading to an accumulation of SlpA in the culture medium of CP23 cells. The present study supports the importance of cell surface structure of L. acidophilus L-92 for SlpA anchoring on the cell surface needed for immunomodulatory effect.





  • Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering in Saccharomyces cerevisiae
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Yu Sasano , Kazuo Yamagishi , Marie Tanikawa , Toshimasa Nakazawa , Minetaka Sugiyama , Yoshinobu Kaneko , Satoshi Harashima

    Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected—a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.





  • Awa1p on the cell surface of sake yeast inhibits biofilm formation and the co-aggregation between sake yeasts and Lactobacillus plantarum ML11-11
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Satoru Hirayama , Masashi Shimizu , Noriko Tsuchiya , Soichi Furukawa , Daisuke Watanabe , Hitoshi Shimoi , Hiroshi Takagi , Hirokazu Ogihara , Yasushi Morinaga

    We examined mixed-species biofilm formation between Lactobacillus plantarum ML11-11 and both foaming and non-foaming mutant strains of Saccharomyces cerevisiae sake yeasts. Wild-type strains showed significantly lower levels of biofilm formation compared with the non-foaming mutants. Awa1p, a protein involved in foam formation during sake brewing, is a glycosylphosphatidylinositol (GPI)-anchored protein and is associated with the cell wall of sake yeasts. The AWA1 gene of the non-foaming mutant strain Kyokai no. 701 (K701) has lost the C-terminal sequence that includes the GPI anchor signal. Mixed-species biofilm formation and co-aggregation of wild-type strain Kyokai no. 7 (K7) were significantly lower than K701 UT-1 (K701 ura3/ura3 trp1/trp1), while the levels of strain K701 UT-1 carrying the AWA1 on a plasmid were comparable to those of K7. The levels of biofilm formation and co-aggregation of the strain K701 UT-1 harboring AWA1 with a deleted GPI anchor signal were similar to those of K701 UT-1. These results clearly demonstrate that Awa1p present on the surface of sake yeast strain K7 inhibits adhesion between yeast cells and L. plantarum ML11-11, consequently impeding mixed-species biofilm formation.





  • Isolation of the stable strain Labrys sp. BK-8 for l(+)-tartaric acid production
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Wenna Bao , Haifeng Pan , Zhenhong Zhang , Yongqing Cheng , Zhipeng Xie , Jianguo Zhang

    A novel cis-epoxysuccinate hydrolase (CESH) producing strain of Labrys sp. BK-8 for production of l(+)-tartaric acid was isolated and identified. After optimization, a maximum activity of 3597 ± 151 U/g was achieved in batch culture in a 10 L fermentor. When Labrys sp. BK-8 was immobilized on κ-carrageenan, the immobilized cells showed a high conversion rate (>99%), enantioselectivity (EE > 99.5%) and storage stability (>90 d). A conversion rate of 97% was still achieved after 10 repeat batches. The CESH was stable over a broad range of temperatures (up to 45°C) and pH values (4.0–10.0). The Labrys sp. BK-8 isolate provides a new alternative with good stability for the industrial biosynthesis of l(+)-tartaric acid.





  • Water-insoluble material from apple pomace makes changes in intracellular NAD+/NADH ratio and pyrophosphate content and stimulates fermentative production of hydrogen
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Osamu Sato , Yuma Suzuki , Yuki Sato , Shinsuke Sasaki , Tomonori Sonoki

    Apple pomace is one of the major agricultural residues in Aomori prefecture, Japan, and it would be useful to develop effective applications for it. As apple pomace contains easily fermentable sugars such as glucose, fructose and sucrose, it can be used as a feedstock for the fermentation of fuels and chemicals. We previously isolated a new hydrogen-producing bacterium, Clostridium beijerinckii HU-1, which could produce H2 at a production rate of 14.5 mmol of H2/L/h in a fed-batch culture at 37 °C, pH 6.0. In this work we found that the HU-1 strain produces H2 at an approximately 20% greater rate when the fermentation medium contains the water-insoluble material from apple pomace. The water-insoluble material from apple pomace caused a metabolic shift that stimulated H2 production. HU-1 showed a decrease of lactate production, which consumes NADH, accompanied by an increase of the intracellular pyrophosphate content, which is an inhibitor of lactate dehydrogenase. The intracellular NAD+/NADH ratios of HU-1 during H2 fermentation were maintained in a more reductive state than those observed without the addition of the water insoluble material. To correct the abnormal intracellular redox balance, caused by the repression of lactate production, H2 production with NADH oxidation must be stimulated.





  • Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Shusuke Okamoto , Taejun Chin , Keisuke Nagata , Tetsuya Takahashi , Hitomi Ohara , Yuji Aso

    Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535T and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli.





  • Microbial resolution of dl-glyceric acid for l-glyceric acid production with newly isolated bacterial strains
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Shun Sato , Tomotake Morita , Tokuma Fukuoka , Dai Kitamoto , Hiroshi Habe

    To produce l-glyceric acid (l-GA) from dl-GA, microbial resolution was investigated using newly isolated bacterial strains capable of enantiospecific degradation of d-GA. Strains GA3R and GA72P, identified as Serratia and Pseudomonas species, respectively, exhausted d-GA within 72 h, resulting in production of l-GA with enantiomeric purity ≥89%.





  • Effects of oral administration of tripeptides derived from type I collagen (collagen tripeptide) on atherosclerosis development in hypercholesterolemic rabbits
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Lihua Tang , Yasuo Sakai , Yoshimichi Ueda , Shogo Katsuda

    Digestion of type I collagen with a collagenase-type protease yields a collagen tripeptide (Ctp) fraction comprising Gly-X-Y sequences that exhibit diverse biological activities. We previously demonstrated that Ctp inhibits the proliferation and migration of cultured aortic smooth muscle cells (SMCs) in vitro. These cells contribute to the pathogenesis of atherosclerosis and other cardiovascular diseases. In order to evaluate the effects of Ctp on atherosclerosis development in vivo, here we used the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit model of familial hypercholesterolemia to determine the effects of oral administration of Ctp for three months. Ctp induced a significant decrease in the area occupied by atherosclerotic plaques in the aorta and in the level of total serum cholesterol. The components of atherosclerotic plaques underwent distinct changes, including reduction in the populations of macrophages and SMCs and a significant decrease in the proportion of macrophages to SMCs. Ctp administration decreased the number of cells in plaques that expressed proliferating cell nuclear antigen and the number of cells with oxidative damage to DNA as indicated by 8-hydroxy-2′-deoxyguanine detection. These findings are the first to define the mechanism underlying the inhibitory effects of Ctp on atherosclerosis development in hypercholesterolemic rabbits, and suggest that Ctp provides an effective therapy for treating atherosclerosis.





  • Effect of oxygen supply on Monascus pigments and citrinin production in submerged fermentation
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Jian Yang , Qi Chen , Weiping Wang , Jiajun Hu , Chuan Hu

    The influence of oxygen supply on Monascus pigments and citrinin production by Monascus ruber HS.4000 in submerged fermentation was studied. For Monascus cultivation with high pigments and low citrinin production, the initial growth phase, mid-stage phase, and later-stage production phase were separated by shifting oxygen supply. The optimal condition for the fermentation process in shake-flask fermentation was a three-stage rotating rate controlled strategy (0–48 h at 150 rpm, 48–108 h at 250 rpm, 108–120 h at 200 rpm) with medium volume of 100 mL added to 250 mL Erlenmeyer flasks at 30°C for 120 h cultivation. Compared to constant one-stage cultivation (medium volume of 100 mL, rotating rate of 250 rpm), the pigments were reduced by 40.4%, but citrinin was reduced by 64.2%. The most appropriate condition for the fermentation process in a 10 L fermentor is also a three-stage aeration process (0–48 h at 300 L/h, 48–96 h at 500 L/h, 96–120 h at 200 L/h) with agitation of 300 rpm at 30°C for 120 h cultivation, and 237.3 ± 5.7 U/mL pigments were produced in 120 h with 6.05 ± 0.19 mg/L citrinin in a 10 L fermentor. Compared to aeration-constant (500 L/h) cultivation, pigment production was increased by 29.6% and citrinin concentration was reduced by 79.5%.





  • Bioprocess of Kosa bioaerosols: Effect of ultraviolet radiation on airborne bacteria within Kosa (Asian dust)
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Fumihisa Kobayashi , Teruya Maki , Makiko Kakikawa , Maromu Yamada , Findya Puspitasari , Yasunobu Iwasaka

    Kosa (Asian dust) is a well-known weather phenomenon in which aerosols are carried by the westerly winds from inland China to East Asia. Recently, the frequency of this phenomenon and the extent of damage caused have been increasing. The airborne bacteria within Kosa are called Kosa bioaerosols. Kosa bioaerosols have affected ecosystems, human health and agricultural productivity in downwind areas. In order to develop a new and useful bacterial source and to identify the source region of Kosa bioaerosols, sampling, isolation, identification, measurement of ultraviolet (UV) radiation tolerance and experimental simulation of UV radiation conditions were performed during Kosa bioaerosol transportation. We sampled these bioaerosols using a Cessna 404 airplane and a bioaerosol sampler at an altitude of approximately 2900 m over the Noto Peninsula on March 27, 2010. The bioaerosol particles were isolated and identified as Bacillus sp. BASZHR 1001. The results of the UV irradiation experiment showed that the UV radiation tolerance of Kosa bioaerosol bacteria was very high compared with that of a soil bacterium. Moreover, the UV radiation tolerance of Kosa bioaerosol spores was higher than that of soil bacterial spores. This suggested that Kosa bioaerosols are transported across the atmosphere as living spores. Similarly, by the experimental simulation of UV radiation conditions, the limited source region of this Kosa bioaerosol was found to be southern Russia and there was a possibility of transport from the Kosa source area.





  • Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Hong-Wei Yen , Jung-Tzu Chang

    The conversion of abundant lignocellulosic biomass (LCB) to valuable compounds has become a very attractive idea recently. This study successfully used LCB (rice straw) hydrolysate as a carbon source for the cultivation of oleaginous yeast-Rhodotorula glutinis in an airlift bioreactor. The lipid content of 34.3 ± 0.6% was obtained in an airlift batch with 60 g reducing sugars/L of LCB hydrolysate at a 2 vvm aeration rate. While using LCB hydrolysate as the carbon source, oleic acid (C18:1) and linoleic acid (C18:2) were the predominant fatty acids of the microbial lipids. Using LCB hydrolysate in the airlift bioreactor at 2 vvm achieved the highest cell mass growth as compared to the agitation tank. Despite the low lipid content of the batch using LCB hydrolysate, this low cost feedstock has the potential of being adopted for the production of β-carotene instead of lipid accumulation in the airlift bioreactor for the cultivation of R. glutinis.





  • Optimization of isopropanol production by engineered cyanobacteria with a synthetic metabolic pathway
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Yasutaka Hirokawa , Iwane Suzuki , Taizo Hanai

    Cyanobacterium is an attractive host for the production of various chemicals and alternative fuels using solar energy and carbon dioxide. In previous study, we succeeded to produce isopropanol using engineered Synechococcus elongatus PCC 7942 under dark and anaerobic conditions (0.43 mM, 26.5 mg/l). In the present study, we report the further optimization of this isopropanol producing condition. We then optimized growth conditions for production of isopropanol by the engineered cyanobacteria, including the use of cells in early stationary phase and buffering of the production medium to neutral pH. We observed that shifting of cultures from dark and anaerobic to light and aerobic conditions during the production phase dramatically increased isopropanol production by conversion to isopropanol from acetate, byproduct under dark and anaerobic condition. Under the optimized production conditions, the titer of isopropanol was elevated 6-fold, to 2.42 mM (146 mg/l).





  • Quantitative analyses of the effect of silk fibroin/nano-hydroxyapatite composites on osteogenic differentiation of MG-63 human osteosarcoma cells
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Linxue Lin , Runsong Hao , Wei Xiong , Jian Zhong

    Silk fibroin (SF)/nano-hydroxyapatite (n-HA) composites are potential biomaterials for bone defect repair. Up to now, the biological evaluation studies of SF/n-HA composites have primarily concentrated on their biocompatibility at cell level such as cell viability and proliferation and tissue level such as material absorption and new bone formation. In this work, SF/n-HA composites were fabricated using a simplified coprecipitation methods and were deposited onto Ti alloy substrates. Then the cell adhesion ability of SF/n-HA composites was observed by SEM and cell proliferation ability of SF/n-HA composites was determined by MTT assay. The ALP activity, BGP contents, and Col I contents of MG-63 human osteosarcoma cells on SF/n-HA composites were quantitatively analyzed. HA nanocrystals were used as controls. These experiments showed that SF/n-HA composites had better cell adhesion and osteogenic differentiation abilities than n-HA materials. This work provides quantitative data to analyze the effect of SF/n-HA composites on cell osteogenic differentiation.





  • Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Akira Ito , Masahiro Yamamoto , Kazushi Ikeda , Masanori Sato , Yoshinori Kawabe , Masamichi Kamihira

    Skeletal muscle regeneration requires migration, proliferation and fusion of myoblasts to form multinucleated myotubes. In our previous study, we showed that insulin-like growth factor (IGF)-I gene delivery stimulates the proliferation and differentiation of mouse myoblast C2C12 cells and promotes the contractile force generated by tissue-engineered skeletal muscles. The aim of this study was to investigate the effects of the extracellular matrix on IGF-I gene-engineered C2C12 cells in vitro. Retroviral vectors for doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into C2C12 cells. When cultured on a type IV collagen-coated surface, we observed significant increases in the migration speed and number of IGF-I gene-engineered C2C12 cells with Dox addition, designated as C2C12/IGF (+) cells. Co-culture of C2C12/IGF (+) cells and parental C2C12 cells, which had been cultured in differentiation medium for 3 days, greatly enhanced myotube formation. Moreover, type IV collagen supplementation promoted the fusion of C2C12/IGF (+) cells with differentiated C2C12 cells and increased the number of myotubes with striations. Myotubes formed by C2C12/IGF (+) cells cultured on type IV collagen showed a dynamic contractile activity in response to electrical pulse stimulation. These findings indicate that type IV collagen promotes skeletal muscle regeneration mediated by IGF-I-expressing myoblasts, which may have important clinical implications in the design of myoblast-based therapies.





  • Development of cytotoxicity-sensitive human cells using overexpression of long non-coding RNAs
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Hidenori Tani , Masaki Torimura

    Biosensors using live cells are analytical devices that have the advantage of being highly sensitive for their targets. Although attention has primarily focused on reporter gene assays using functional promoters, cell viability assays are still efficient. We focus on long non-coding RNAs (lncRNAs) that are involved in the molecular mechanisms associated with responses to cellular stresses as a new biological material. Here we have developed human live cells transfected with lncRNAs that can be used as an intelligent sensor of cytotoxicity for a broad range of environmental stresses. We identified three lncRNAs (GAS5, IDI2-AS1, and SNHG15) that responded to cycloheximide in HEK293 cells. Overexpression of these lncRNAs sensitized human cells to cell death in response to various stresses (cycloheximide, ultraviolet irradiation, mercury II chloride, or hydrogen peroxide). In particular, dual lncRNA (GAS5 plus IDI2-AS1, or GAS5 plus SNHG15) overexpression sensitized cells to cell death by more cellular stresses. We propose a method for highly sensitive biosensors using overexpression of lncRNAs that can potentially measure the cytotoxicity signals of various environmental stresses.





  • Controlled processing of a full-sized porcine liver to a decellularized matrix in 24 h
    Publication date: May 2015
    Source:Journal of Bioscience and Bioengineering, Volume 119, Issue 5

    Author(s): Nicola Elena Maria Bühler , Klaus Schulze-Osthoff , Alfred Königsrainer , Martin Schenk

    The generation of full-sized humanized organs based on animal matrix scaffolds is a promising approach to overcome the shortage of transplant organs. Recent decellularization methods are mostly time-consuming and associated with large rinsing volumes and poorly standardized procedures. In this study we developed an optimized rapid and standardized decellularization method to obtain a functional porcine liver matrix within 24 h. Full porcine livers (n = 10) were decellularized by flushing with 3 L of an isotonic sodium chloride solution and controlled portal perfusion (20 mmHg) with 2 × 10 L of a 1% sodium dodecyl sulphate (SDS) solution at 37°C and a final perfusion with DNase (n = 5). Protein concentrations were continuously monitored by optical density (280 nm). DNA, glycosaminoglycans, and collagen contents were assessed and a haematoxylin and eosin (H&E) staining was performed. After 24 h of perfusion, the liver had a white and translucent appearance, and no further protein was eluted. Histological staining showed an intact extracellular matrix with no nuclear residuals. Moreover, only trace amounts of DNA were detectable in the decellularized tissue (p < 0.001), while glycosaminoglycans and about 60% of collagen levels could be preserved. Thus, we demonstrate that human-scale porcine livers can be successfully decellularized with small volumes of an SDS solution and DNase in a standardized process within 24 h to obtain a clinically relevant organ scaffold suitable for further tissue engineering.