Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 76 , Issue 7
Showing 1-31 articles out of 31 articles from the selected issue
Award Reviews
  • Hiroyuki KONNO
    2012 Volume 76 Issue 7 Pages 1257-1261
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Synthetic studies of Annonaceous acetogenins isolated from the plant family Annonaceae and marine natural products (miraziridine A, tokaramide A, and callipeltins) containing unusual amino acids, and recent studies of the structure-activity relationship of cysteine protease inhibitor are described.
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  • Yu NAKAGAWA
    2012 Volume 76 Issue 7 Pages 1262-1274
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.
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Analytical Chemistry Regular Papers
  • Katsumi HASHIZUME, Toshihiko ITO, Misako SHIMOHASHI, Atsufumi KOKITA, ...
    2012 Volume 76 Issue 7 Pages 1291-1295
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.
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  • Rei YAMAKAWA, Yoshiko TAKUBO, Kanako OHBAYASHI, Hideshi NAKA, Tetsu AN ...
    2012 Volume 76 Issue 7 Pages 1303-1307
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The plum cankerworm moth, Cystidia couaggaria couaggaria (Geometridae: Ennominae), is a defoliator of Chinese plum trees (Prunus mume). The pheromone components of the female were analyzed by gas chromatography (GC) with an electro-antennographic (EAG) detector and GC coupled with mass spectrometry. The crude pheromone extract included several EAG-active components, i.e., trienyl, dienyl, and saturated hydrocarbons, with a C21–C25 straight chain. The characteristic mass spectra indicated the unsaturated hydrocarbons to be (3Z,6Z,9Z)-3,6,9-trienes and (6Z,9Z)-6,9-dienes. In the fields, mixtures of the synthetic C21 and C23 trienes in a ratio of 2:3 and 1:4 successfully attracted males of this diurnal species during daytime. While the male antennae responded to the C25 triene and saturated hydrocarbons, their synergistic effects were not observed on the male attraction in the fields. Addition of the C21 diene interestingly inhibited the activity of the triene mixture. Males of Cystidia truncangulata, a sympatric diurnal congener of C. c. couaggaria, showed similar EAG responses to the unsaturated hydrocarbons, but no C. truncangulata males were attracted by the lures tested for C. c. couaggaria males, indicating that the identified hydrocarbons comprised the species-specific pheromone of C. c. couaggaria females.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Mitsuki FUKUMOTO, Daizou KUDOU, Shouko MURANO, Tomoo SHIBA, Dan SATO, ...
    2012 Volume 76 Issue 7 Pages 1275-1284
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.
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  • Jung-Min YOO, Ji-Hee KIM, Sae-Jin PARK, Yeo-Jin KANG, Tack-Joong KIM
    2012 Volume 76 Issue 7 Pages 1285-1290
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Mast cells are the principal effector cells involved in the allergic response, through the release of histamine. We investigated the effect of eriodictyol, derived from the painted maple and yerba santa, on mast cell degranulation and on an allergic response in an animal model. We also investigated its effect on the expression of the ceramide kinase (CERK) involved in calcium-dependent degranulation, and on ceramide activation by multiple cytokines. Eriodictyol suppressed the release of beta-hexosaminidase, a marker of degranulation, and the expression of interleukin (IL)-4 mRNA. It inhibited the expression of CERK mRNA, reduced the ceramide concentration in antigen-stimulated mast cells, and suppressed the passive cutaneous anaphylaxis (PCA) reaction in mice in a dose-dependent manner. These results suggest that eriodictyol can inhibit mast cell degranulation through inhibition of ceramide kinase, and that it might potentially serve as an anti-allergic agent.
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  • Sugunya PITAKRATTANANUKOOL, Taiji KAWAKATSU, Somboon ANUNTALABHOCHAI, ...
    2012 Volume 76 Issue 7 Pages 1296-1302
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Supplementary material
    Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress–inducible gene that plays an important role in the leaf senescence process.
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  • Joo-Hyun SEO, Joon-Young HWANG, Su-Hyun SEO, Hyunjong KANG, Bum-Yeol H ...
    2012 Volume 76 Issue 7 Pages 1308-1314
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Supplementary material
    ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appreared to be determined by residues positioned at the entrance region of the active site binding pockets.
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  • Kimio UEMATSU, Nobuaki SUZUKI, Tomoko IWAMAE, Masayuki INUI, Hideaki Y ...
    2012 Volume 76 Issue 7 Pages 1315-1321
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Plastidial phosphoglucomutase (PGM) plays an important role in starch synthesis and degradation. Nonetheless, the impact of enhanced plastidial PGM activity on metabolism in photosynthetic tissue is yet to be elucidated. In this study, we generated transplastomic tobacco plants overproducing Arabidopsis thaliana plastidial PGM (AtptPGM) in chloroplasts and analyzed the consequent metabolic and physiological parameters in the transplastomic plants. AtptPGM accumulated in the chloroplasts to up to 16% of total soluble protein in the leaves. PGM activity in leaves increased 100-fold relative to that of wild-type plants. The transplastomic plants were phenotypically indistinguishable in their growth rates, photosynthetic activities, and starch synthesis from wild-type plants, but hexose partitioning in the light period was dramatically different. Furthermore, alteration of extracellular invertase activity was observed in the lower leaves of the transplastomic plants. These observations suggest that high-level expression of plastidial PGM alters hexose partitioning in light periods via modification of extracellular invertase activity.
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  • Chaoqing HUANG, Xiaoguang CHEN, Baolin GUO, Wenhua HUANG, Ting SHEN, X ...
    2012 Volume 76 Issue 7 Pages 1322-1328
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The anti-tumor effect of Icariside II (IcaS), a natural prenylated flavonol glycoside, was studied on human breast cancer MCF7 cells to unveil the underlying mechanisms involved. IcaS in MCF7 cells produced a loss of mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9 revealed the involvement of the intrinsic apoptosis pathway. In contrast, IcaS enhanced the expression level of Fas and the Fas-associated death domain (FADD), and activated caspase-8, suggesting the involvement of the extrinsic apoptosis pathway. IcaS also increased the expression of Bax and BimL without affecting the expression status of Bcl-2 and Bid, suggesting that the apoptosis induced by IcaS was related to Bcl-2 family protein regulation. IcaS thus induced apoptosis in MCF7 cells involving both the intrinsic and extrinsic signaling pathways. Its potential as a candidate for an anti-cancer agent warrants further investigation.
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  • Miyuki YOKORO, Makiko SUZUKI, Kaeko MUROTA, Chie OTSUKA, Hiromi YAMASH ...
    2012 Volume 76 Issue 7 Pages 1334-1342
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    NG,NG-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.
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  • Xiao-Tao FENG, Tian-Zhan WANG, Jing LENG, Yi CHEN, Ji-Bo LIU, Yi LIU, ...
    2012 Volume 76 Issue 7 Pages 1356-1361
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.
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  • Makoto OGATA, Ryota TAKEUCHI, Akari SUZUKI, Hirofumi HIRAI, Taichi USU ...
    2012 Volume 76 Issue 7 Pages 1362-1366
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2–4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GNnL) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN3L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN2L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6–54.3% at a concentration of 400 µM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.
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  • Sa-sa GU, Yan ZHANG, Xiao-li LI, Shu-yan WU, Teng-yue DIAO, Rong HAI, ...
    2012 Volume 76 Issue 7 Pages 1367-1371
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The local tissue-specific renin-angiotensin system (RAS) was identified. The aim of this study was to investigate the role of local bone RAS in the osteoporosis of aging mice. Twelve-month-old and two-month-old male mice were respectively assigned to the ageing and young groups. The tibias and femurs were collected for an analysis of histomorphology, bone mass, and gene and protein expression. H&E staining and micro-CT measurement showed a loss of the trabecular bone network and decrease of bone mineral density in the proximal tibial metaphysis of the aged mice. The PCR results indicated the significant up-regulation of renin and angiotensinogen (AGT) mRNA expression in both the tibia and femur of the ageing mice. Western blotting data showed that the tibial angiotensin II protein expression was significantly increased in the ageing group. The enhancement of renin and AGT expression in the bone tissue resulted in the increased production of angiotensin II which plays an important role in the pathology of age-related osteoporosis.
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  • Tomokazu FUKUDA, Masafumi KATAYAMA, Takayuki YOSHIZAWA, Takahiro EITSU ...
    2012 Volume 76 Issue 7 Pages 1372-1377
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.
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  • Dae Hoon KIM, Naoki MORIMOTO, Wataru SABURI, Atsushi MUKAI, Koji IMOTO ...
    2012 Volume 76 Issue 7 Pages 1378-1383
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4–10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.
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  • Ryosuke MUNAKATA, Tsuyoshi INOUE, Takao KOEDUKA, Kanako SASAKI, Yusuke ...
    2012 Volume 76 Issue 7 Pages 1389-1393
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Supplementary material
    Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent Km values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Yuko SATO, Tsudoi TOYODA, Akiko SHIMIZU-IBUKA, Tomoko TAMURA, Kazuo KO ...
    2012 Volume 76 Issue 7 Pages 1329-1333
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 µM against ACE and may have potential use as a functional food.
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  • Sayuri TSURUDA, Kouichi AKAKI, Hiroshi HIWAKI, Akira SUZUKI, Hiroshi A ...
    2012 Volume 76 Issue 7 Pages 1343-1349
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.
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  • Masaru OCHIAI, Tatsuhiro MATSUO
    2012 Volume 76 Issue 7 Pages 1350-1355
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    Up-regulation of stearoyl-CoA desaturase (SCD) is closely related to improved insulin resistance. We investigated whether the SCD indices in tissues were influenced by a single-endurance exercise and low content of dietary medium-chain fatty acid (FA). Wistar rats were fed a long-chain (S) or medium- and long-chain FA (M) diet for 2 weeks. At the end of the experiment, the rats were further assigned to two sub-groups (sedentary, Sed; exercise, Ex). These groups were defined as S-Sed, S-Ex, M-Sed, and M-Ex. The rats in the exercise groups were subjected to swimming exercise for 4 h, and tissue samples were obtained. The exercise significantly increased the triglyceride (TG) content and SCD index only in the liver. In contrast, no such changes were apparent by intake of the M diet. A single bout of endurance exercise increased the hepatic TG content and SCD index which might be effective in protecting against insulin resistance.
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Food & Nutrition Science Note
Microbiology & Fermentation Technology Regular Paper
  • Wen-Rong XIA, Wen-Liang FU, Ling CAI, Xin CAI, Yuan-Yuan WANG, Min-Ji ...
    2012 Volume 76 Issue 7 Pages 1384-1388
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.
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Microbiology & Fermentation Technology Communication
  • Ken-ichi HASHIMOTO, Jun MURATA, Tomoyuki KONISHI, Isamu YABE, Tsuyoshi ...
    2012 Volume 76 Issue 7 Pages 1422-1424
    Published: July 23, 2012
    Released: July 23, 2012
    [Advance publication] Released: July 07, 2012
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    The NCgl1221 gene, which encodes a mechanosensitive channel, has been reported to be critically involved in glutamate (Glu) overproduction by Corynebacterium glutamicum, but direct evidence of Glu excretion through this channel has not yet been provided. In this study, by electrophysiological methods, we found direct evidence of Glu excretion through this channel by passive diffusion. We found that the introduction into Phe-producing Escherichia coli of mutant NCgl1221 genes that induce Glu overproduction by C. glutamicum improved productivity. This suggests a low-substrate preference of this channel, indicates its potential as a versatile exporter, and more broadly, indicates the potential of exporter engineering.
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