JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.117 cover

 

Journal of Bioscience and Bioengineering – Recent Articles

  • Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Saiko Sugawara , Toshihiko Ito , Shiori Sato , Yuki Sato , Kano Kasuga , Ikuo Kojima , Masayuki Kobayashi

    In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro31–Leu202) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser50 and Leu51 was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu51–Leu202), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.





  • Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Yusuke Kawano , Midori Sekine , Masaki Ihara

    Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the k cat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.





  • Arginine and lysine reduce the high viscosity of serum albumin solutions for pharmaceutical injection
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Naoto Inoue , Eisuke Takai , Tsutomu Arakawa , Kentaro Shiraki

    Therapeutic protein solutions for subcutaneous injection must be very highly concentrated, which increases their viscosity through protein–protein interactions. However, maintaining a solution viscosity below 50 cP is important for the preparation and injection of therapeutic protein solutions. In this study, we examined the effect of various amino acids on the solution viscosity of very highly concentrated bovine serum albumin (BSA) and human serum albumin (HSA) at a physiological pH. Among the amino acids tested, l-arginine hydrochloride (ArgHCl) and l-lysine hydrochloride (LysHCl) (50–200 mM) successfully reduced the viscosity of both BSA and HSA solutions; guanidine hydrochloride (GdnHCl), NaCl, and other sodium salts were equally as effective, indicating the electrostatic shielding effect of these additives. Fourier transform infrared spectroscopy showed that BSA is in its native state even in the presence of ArgHCl, LysHCl, and NaCl at high protein concentrations. These results indicate that weakened protein–protein interactions play a key role in reducing solution viscosity. ArgHCl and LysHCl, which are also non-toxic compounds, will be used as additives to reduce the solution viscosity of concentrated therapeutic proteins.





  • Purification and characterization of a novel fibrinolytic α chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Sung-Min Moon , Jae-Sung Kim , Heung-Joong Kim , Mi Suk Choi , Bo Ram Park , Su-Gwan Kim , Hoon Ahn , Hong Sung Chun , Yong Kook Shin , Jong-Jin Kim , Do Kyung Kim , Sook-Young Lee , Young-Woo Seo , Yong Hwan Kim , Chun Sung Kim

    A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21 kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37°C, respectively. Enzymatic activity was inhibited by Cu2+ and Co2+. It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5 μg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bβ- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease.





  • Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Sadanobu Fukuyama , Hisaaki Mihara , Ryoma Miyake , Makoto Ueda , Nobuyoshi Esaki , Tatsuo Kurihara

    2,4-Diaminopentanoate dehydrogenase (2,4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD+- or NADP+-dependent oxidative deamination of (2R,4S)-2,4-diaminopentanoate (2,4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2,4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD+ and NADP+ in the presence of 2,4-DAP, indicating that the protein is a 2,4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2,4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)+-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2,4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2,4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2,4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2,4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis–Menten kinetics toward 2,5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent K i value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway.





  • Production of antioomycete compounds active against the phytopathogens Phytophthora sojae and Aphanomyces cochlioides by clavicipitoid entomopathogenic fungi
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Sastia Prama Putri , Kei-ichi Ishido , Hiroshi Kinoshita , Shigeru Kitani , Fumio Ihara , Yasuko Sakihama , Yasuhiro Igarashi , Takuya Nihira

    A total of 412 strains belonging to 14 genera of clavicipitoid entomopathogenic fungi (EPF) were screened for activities against two economically important plant pathogenic oomycetes, Phytophthora sojae and Aphanomyces cochlioides. To identify the antioomycete compounds produced by EPF, the extracts of 13 highly active EPF strains were characterized in detail by high performance liquid chromatography with diode array detection and high-resolution mass spectrometric detection and antioomycete assay. The antioomycete activity of several Metarhizium extracts was associated with previously isolated aurovertins, fungerin, N-(methyl-3-oxodec-6-enoyl)-2-pyrroline, and N-(methyl-3-oxodecanoyl)-2-pyrroline. The depsipeptide beauvericin was confirmed to be one of the active principles of three strains of Isaria tenuipes, which strongly inhibited mycelial growth of both P. sojae and A. cochlioides. Two known bioactive metabolites, paecilosetin and aranorosinol A, together with a novel and potent antioomycete compound, farinomalein, were isolated from the extracts of Isaria farinosa and all compounds were confirmed to have antioomycete activity. Identification of 8 antioomycete compounds from 13 clavicipitioid EPF demonstrated a new potential use of EPF as a source of compounds for the control of soil-borne plant pathogenic oomycetes.





  • Functional analysis of cis-aconitate decarboxylase and trans-aconitate metabolism in riboflavin-producing filamentous Ashbya gossypii
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Takashi Sugimoto , Tatsuya Kato , Enoch Y. Park

    In Ashbya gossypii, isocitrate lyase (ICL1) is a very crucial enzyme for riboflavin production. Itaconate, the inhibitor of ICL1, has been used as an antimetabolite for mutagenic studies in A. gossypii. It has been reported that itaconate is produced from cis-aconitate by cis-aconitate decarboxylase (CAD1) in Aspergillus terreus. In this study, identification of CAD1 gene and determination of the presence of itaconate in the riboflavin biosynthetic pathway in A. gossypii were carried out to confirm itaconate metabolism. Although no CAD1 candidate gene was found and no itaconate production was observed, cis- and trans-aconitate were detected in the riboflavin production phase. It is known that trans-aconitate inhibits aconitase (ACO1) in the tricarboxylic acid cycle. In A. gossypii, the transcription level of AGR110Wp, the homolog of trans-aconitate 3-methyltransferase (TMT1), was enhanced by almost threefold during riboflavin production than that during its growth phase. TMT1 catalyzes the methylation reaction of trans-aconitate in Saccharomyces cerevisiae. Thus, these results suggest that the enhancement of the transcription level of this TMT1 homolog decreases the trans-aconitate level, which may mitigate the inhibition of ACO1 by oxidative stress in the riboflavin biosynthetic pathway in A. gossypii. This is a novel finding in A. gossypii, which may open new metabolic engineering ideas for improving riboflavin productivity.





  • Evaluation of microbial diversity in sulfite-added and sulfite-free wine by culture-dependent and -independent methods
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Masayuki Takahashi , Tami Ohta , Kazuo Masaki , Akihiro Mizuno , Nami Goto-Yamamoto

    The difference in microbiota including non-lactic acid bacteria, non-acetic acid bacteria, and wild yeast during winemaking and in the end-products between sulfite-added and sulfite-free wine, was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and a culture-dependent method. There were differences between the microorganisms detected by PCR-DGGE and those detected by the culture-dependent method, probably because of the selectivity of culture medium and the characteristics of PCR-based method. In both the red wine and white wine, the microbial diversity of the sulfite-added wine was lower than that of the sulfite-free wine during fermentation. Tatumella terrea was detected from the fermenting must by PCR-DGGE and by the culture-dependent method, even though sulfite inhibited its growth to some extent. We confirmed that the addition of sulfite plays an important role in winemaking by inhibiting the growth of unexpected microorganisms, but on the other hand, it was revealed that some microorganisms can survive and grow in sulfite-added fermenting must. We also analyzed 15 samples of commercial wines by the PCR-DGGE method and detected various microorganisms. Among them, Sphingomonas sp., Pseudozyma sp., Ochromonas sp. and Methylophilus sp. were found for the first time in wine as far as we know. We did not identify a specific microorganism that was detected only from wines without sulfite addition. Thus, the microbiota of end-products seemed to be influenced by other factors, such as filtration before bottling, the production equipment and the storage environment.





  • Isolation of a selenite-reducing and cadmium-resistant bacterium Pseudomonas sp. strain RB for microbial synthesis of CdSe nanoparticles
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Hiroyuki Ayano , Masaki Miyake , Kanako Terasawa , Masashi Kuroda , Satoshi Soda , Toshifumi Sakaguchi , Michihiko Ike

    Bacteria capable of synthesizing CdSe from selenite and cadmium ion were enriched from a soil sample. After repeated transfer of the soil-derived bacterial cultures to a new medium containing selenite and cadmium ion 42 times (during 360 days), an enrichment culture that can simultaneously remove selenite and cadmium ion (1 mM each) from the liquid phase was obtained. The culture's color became reddish-brown, indicating CdSe nanoparticle production, as confirmed by energy-dispersive x-ray spectra (EDS). As a result of isolation operations, the bacterium that was the most responsible for synthesizing CdSe, named Pseudomonas sp. RB, was obtained. Transmission electron microscopy and EDS revealed that this strain accumulated nanoparticles (10–20 nm) consisting of selenium and cadmium inside and on the cells when cultivated in the same medium for the enrichment culture. This report is the first describing isolation of a selenite-reducing and cadmium-resistant bacterium. It is useful for CdSe nanoparticle synthesis in the simple one-vessel operation.





  • Effect of salinity on N2O production during shortcut biological nitrogen removal from landfill leachate
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Mu Liu , Tiantian Liu , Yongzhen Peng , Shuying Wang , Han Xiao

    Three identical SBR adapted to different salinity were applied to investigate the characteristics of the treatment performance and N2O production ( P N 2 O ) during shortcut biological nitrogen removal from landfill leachate under various operating parameters. Increase of salinity might deteriorate the activity of the microorganisms leading to the increase of P N 2 O , however, the system could be gradually adapted to the inhibition and alleviate the detrimental effect to some extent. The system acclimated to high salinity provided better performance under high salinity shock and a lower possibility of P N 2 O , while a sudden decrease in salinity can cause a temporary increase in P N 2 O . High salinity strengthened the influence of high ammonia nitrogen concentration and low DO concentration on P N 2 O while the strengthening effect was unconspicuous at high DO concentration. The anoxic phase did not produce a significant amount of N2O even at the lowest C/N ratio of 0.5 and was less susceptible to salinity. Characterization of the biomass composition using fluorescence in situ hybridization analysis confirmed that the relative proportion of Nitrosomonas europaea was increased with the increase of the salinity, which may be an important factor for the strengthening effect of salinity on P N 2 O .





  • Direct proteomic mapping of Streptomyces roseosporus NRRL 11379 with precursor and insights into daptomycin biosynthesis
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Chiming Ye , I-Son Ng , Keju Jing , Yinghua Lu

    This first-attempt study provided liquid chromatography tandem mass (LC–MS/MS) proteomics approach to explore precursor effects on daptomycin synthesis from Streptomyces roseosporus NRRL 11379. Among all, 357 and 691 differential proteins from 601 proteins in precursor group (144 h+) and 935 proteins in non-precursor group (144 h−) were identified, respectively. Through the simulation of the 2D-protein mapping, most proteins were found in isoelectric points ranged of 4.5–10.0 as well as Mws ranged 10–100 kDa. As a result, LC–MS/MS analysis was consistence with the analytical results of two-dimensional electrophoresis (2DE) but provided much intact profiles of proteins by precursor effect on S. roseosporus. To have more insight exploration, differential proteins associated to Streptomyces spp. were defined into 14 groups of their functional classification. The major differential proteins were in transport/membrane functional group with an occupation of 12.4% for 144 h+ and 5.2% for 144 h−, respectively. LC–MS/MS results as a direct proteomic mapping approach reveal more daptomycin synthetic and regulation-related proteins from precursor group in terms of methyltransferase, ATP-binding cassette (ABC) transporters, resistance proteins and regulators.





  • Increase in lactate yield by growing Corynebacterium glutamicum in a bioelectrochemical reactor
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Kengo Sasaki , Yota Tsuge , Daisuke Sasaki , Akihiko Kondo

    Under conditions conductive to growth, Corynebacterium glutamicum showed higher lactate yield from glucose (1.62 ± 0.04) in a bioelectrochemical reactor including 0.2 mM of anthraquinone 2,6-disulfonate with the electrode potential regulated at −0.6 V (vs. Ag/AgCl) than in a non-regulated environment (1.10 ± 0.03), clarifying that low cathodic potential is beneficial for lactate production.





  • Absorption and metabolism of formaldehyde in solutions by detached banana leaves
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Zhidong Zeng , Chuanjiao Qi , Qi Chen , Kunzhi Li , Limei Chen

    Detached banana leaves are one of the by-products of banana production. In this study, the absorption and metabolism of formaldehyde (HCHO) in solutions by detached banana leaves was investigated under submergence conditions. The results showed that banana leaves could effectively absorb HCHO in the treatment solutions, and the relationship between HCHO absorption and treatment time appeared to fit a radical root function model. 13C nuclear magnetic resonance analysis was used to investigate the ability of detached banana leaves to metabolise H13CHO, and the results indicated that the H13CHO absorbed from the treatment solutions was converted into non-toxic compounds. High amounts of [U-13C]glucose, [U-13C]fructose, [3-13C]serine and [3-13C]citrate were produced as a result of H13CHO metabolism in banana leaves, and the production of a small amount of [2,4-13C]citrate and [2,3-13C]alanine was also observed. These results suggest that detached banana leaves can metabolise H13CHO and convert it to non-toxic compounds. The metabolic pathways that produce these intermediates in detached banana leaves are postulated based on our 13C nuclear magnetic resonance data.





  • Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Fantao Kong , Tomohito Yamasaki , Takeshi Ohama

    We attempted to overexpress three types of expression cassettes, each of which contained a different open reading frame (ORF) of domestic Chlamydomonas cDNAs. Each ORF was strongly driven by an artificial hybrid promoter. We used two wild-type Chlamydomonas strains (i.e., CC-124 and CC-125) and two mutant strains [i.e., UV-mutated (UVM) 4 and UVM11] that have been reported to have a high potency for expressing nondomestic nuclear transgenes. We found that the 1-deoxy-d-xylulose-5-phosphatesynthase (DXS1), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR1), and squalene synthase (SQS) cassettes were not readily overexpressed in the wild-type strains at levels where the products were clearly detectable by Western blotting using a monoclonal antibody. In contrast, Western blot-positive SQS cassette transformants were frequently detected in the UVM4 and UVM11 strains, i.e., at an approximately 4.5 times higher frequency than that in the CC-124 wild-type strain. Moreover, transformants that accumulated large amounts of the SQS protein were obtained frequently in the UVM4 and UVM11 strains, i.e., the frequency was approximately 2.2 times higher than that in the CC-124 strain. However, a position effect of the integrated expression cassette was obviously detected not only in the wild-type but also in UVM strains. This suggests that the epigenetic repression mechanism of transgenic genes was not completely knocked out, even in the UVM strains. Further improved Chlamydomonas strains are essential to facilitate high-throughput screening of transformants that express nuclear transgenes at a high level.





  • Culture of a high-chlorophyll-producing and halotolerant Chlorella vulgaris
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Koichi Nakanishi , Keiji Deuchi

    In order to increase the value of freshwater algae as raw ingredients for health foods and feed for seawater-based farmed fish, we sought to breed high-chlorophyll halotolerant Chlorella with the objective of generating strains with both high chlorophyll concentrations (≥5%) and halotolerance (up to 1% NaCl). We used the Chlorella vulgaris K strain in our research institute culture collection and induced mutations with UV irradiation and acriflavine which is known to effect mutations of mitochondrial DNA that are associated with chlorophyll production. Screenings were conducted on seawater-based “For Chlorella spp.” (FC) agar medium, and dark-green-colored colonies were visually selected by macroscopic inspection. We obtained a high-chlorophyll halotolerant strain (designated C. vulgaris M-207A7) that had a chlorophyll concentration of 6.7% (d.m.), a level at least three-fold higher than that of K strain. This isolate also exhibited a greater survival rate in seawater that of K strain.





  • Cis-element of the rice PDIL2-3 promoter is responsible for inducing the endoplasmic reticulum stress response
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Hideyuki Takahashi , Shuyi Wang , Shimpei Hayashi , Yuhya Wakasa , Fumio Takaiwa

    A protein disulfide isomerase (PDI) family oxidoreductase, PDIL2-3, is involved in endoplasmic reticulum (ER) stress responses in rice. We identified a critical cis-element required for induction of the ER stress response. The activation of PDIL2-3 in response to ER stress strongly depends on the IRE1-OsbZIP50 signaling pathway.





  • Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Baiyao Xu , Yang Ju , Guanbin Song

    Adipogenesis is important to health and is thought occurring in the two stages of mesenchymal stem cell commitment to a preadipocyte fate and terminal differentiation of the preadipocyte. However, the mechanism of adipogenesis is still not clear. In this study, the roles of p38, extracellular regulated protein kinases 1/2 (ERK1/2), focal adhesion kinase (FAK), RhoA/ROCK, and cytoskeleton in both of the two stages of adipogenesis were assayed. Our results showed that the treatments of SB203580 (the inhibitor of p38) and U0126 (the inhibitor of ERK1/2) suppressed the adipogenesis induced by differentiation medium, and the treatments of PF573228 (a specific inhibitor of FAK), Y27632 (a specific inhibitor of RhoA/ROCK) and cytochalasin D (an inhibitor of cytoskeletal organization) promoted the adipogenesis. The treatments of SB203580 and U0126 significantly inhibited the adipogenic differentiation of hMSCs cultured in differentiation medium in the presence of PF573228, Y27632 or cytochalasin D. Moreover, the treatments of PF573228, Y27632 and cytochalasin D promoted p38 and ERK1/2 phosphorylations, and the treatments of U0126 and SB203580 decreased p38 and ERK1/2 phosphorylations, respectively. These results demonstrated that p38 and ERK1/2 played crucial positive roles in adipogenesis, and FAK, RhoA/ROCK and cytoskeleton played negative roles. Furthermore, FAK, RhoA/ROCK and cytoskeleton affected adipogenesis by regulating the activities of p38 and ERK1/2 which interacted with each other in the process of adipogenesis.





  • Trehalose suppresses antibody aggregation during the culture of Chinese hamster ovary cells
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Masayoshi Onitsuka , Miki Tatsuzawa , Ryutaro Asano , Izumi Kumagai , Akihiro Shirai , Hideaki Maseda , Takeshi Omasa

    The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies.





  • Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Masayoshi Onitsuka , Akira Kawaguchi , Ryutaro Asano , Izumi Kumagai , Kohsuke Honda , Hisao Ohtake , Takeshi Omasa

    N-Glycosylation of therapeutic antibodies contributes not only to their biological function, but also to their stability and tendency to aggregate. Here, we investigated the impact of the glycosylation status of an aggregated antibody that accumulated during the bioreactor culture of Chinese hamster ovary cells. High-performance liquid chromatography analysis showed that there was no apparent difference in the glycosylation patterns of monomeric, dimeric, and large aggregated forms of the antibody. In contrast, lectin binding assays, which enable the total amounts of specific sugar residues to be detected, showed that both galactose and fucose residues in dimers and large aggregates were reduced to 70–80% of the amount in monomers. These results strongly suggest that the lack of N-linked oligosaccharides, a result of deglycosylation or aglycosylation, occurred in a proportion of the dimeric and large aggregated components. The present study demonstrates that glycosylation heterogeneities are a potential cause of antibody aggregation in cell culture of Chinese hamster ovary cells, and that the lack of N-glycosylation promotes the formation of dimers and finally results in large aggregates.





  • Selective responses of enzymes in the two parallel pathways of rosmarinic acid biosynthetic pathway to elicitors in Salvia miltiorrhiza hairy root cultures
    Publication date: May 2014
    Source:Journal of Bioscience and Bioengineering, Volume 117, Issue 5

    Author(s): Shuncang Zhang , Yan Yan , Bangqing Wang , Zongsuo Liang , Yan Liu , Fenghua Liu , Zhihong Qi

    Rosmarinic acid and salvianolic acid B are two important phenolic compounds with therapeutic properties in Salvia miltiorrhiza Bunge. The biosynthesis of rosmarinic acid is initiated by two parallel pathways, namely the phenylpropanoid pathway and the tyrosine-derived pathway. Salvianolic acid B is a structural dimer of rosmarinic acid and is believed to be derived from rosmarinic acid. In the current study, methyl jasmonate (MeJA) and hyphal extracts from fungi were used as elicitors to examine the relationship between enzymes in the two parallel pathways and accumulation of phenolic compounds in S. miltiorrhiza hairy root cultures. The results showed that accumulations of rosmarinic acid, salvianolic acid B and total phenolics were enhanced by MeJA while suppressed by fugal extracts. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitors treated. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to rosmarinic acid and salvianolic acid B biosynthesis in S. miltiorrhiza hairy roots.