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991.
Choi HM Chang JY Trinh le A Padilla JE Fraser SE Pierce NA 《Nature biotechnology》2010,28(11):1208-1212
In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization. 相似文献
992.
Jung Min Song Kiwoong Nam Young-Uk Sun Mee Hye Kang Choong-Gon Kim Suk-Tae Kwon Jehee Lee Youn-Ho Lee 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2010,155(4):403-412
Collembolan species have been known to have β-1,3-glucanase activity and yet the genes coding such enzymes have not been demonstrated. We report here a novel arthropod endo-β-1,3-glucanase gene CaLam from the Antarctic springtail, Cryptopygus antarcticus. The open reading frame consists of 813 bp encoding 270 amino acids with a putative signal peptide and a typical motif of glycosyl hydrolase family 16 (GHF16), E–I–D–I–T–E. The recombinant protein expressed in E. coli shows the hydrolytic activity toward laminarin (Km ~ 9.98 mg/mL) with an optimal temperature 50 °C and an optimal pH 6.0. CaLam digests laminarin and laminarioligosaccharides except laminaribiose as an endo-β-1,3-glucanase, releasing glucose, laminaribiose and laminaritriose as the major products. Analyses of molecular phylogeny of CaLam and its protein structure reveal that CaLam is closely related with bacterial β-1,3-glucanases more than with the eukaryotic homologues. Even so, the genomic structure of the CaLam gene consisting of six exons interspersed with approximately 57 to 63 bp introns confirms that it is endogenous in the genome of the Antarctic springtail. These results suggest that CaLam should have been transferred from bacteria to the lineage of the Collembolan species by horizontal gene transfer. 相似文献
993.
Assessment of soil fungal communities using pyrosequencing 总被引:1,自引:0,他引:1
Young Woon Lim Byung Kwon Kim Changmu Kim Hack Sung Jung Bong-Soo Kim Jae-Hak Lee Jongsik Chun 《Journal of microbiology (Seoul, Korea)》2010,48(3):284-289
Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in
soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived
from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number
of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and
95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa
were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa
in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have
been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including
species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales,
Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high
diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities
in soils. 相似文献
994.
Selvaraj T Kim SK Kim YH Jeong YS Kim YJ Phuong ND Jung KH Kim J Yun HD Kim H 《Journal of microbiology (Seoul, Korea)》2010,48(6):856-861
A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding. 相似文献
995.
Subramanian Mohan Raj Chelladurai Rathnasingh Woo-Chel Jung Edwardraja Selvakumar Sunghoon Park 《Biotechnology and Bioprocess Engineering》2010,15(1):131-138
3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological
production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary
compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad
substrate specificity for various aliphatic aldehydes, especially C2–C5 aldehydes. NAD+ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature
for PuuC activity were pH 8.0 and 45°C. The K
m values for 3-HPA and NAD+ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol
or dithiothreitol, while several metal ions, particularly Hg2+, Ag+, and Cu2+ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the
glycine motif, suggesting that PuuC belongs to class 2 ALDHs. 相似文献
996.
Mee‐Sun Hyun Jung‐Mu Hur Yeun‐Ja Mun Dongho Kim Won‐Hong Woo 《Journal of cellular biochemistry》2010,109(2):329-338
Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
997.
Sarah E. Ross Alan R. Mardinly Alejandra E. McCord Jonathan Zurawski Sonia Cohen Cynthia Jung Linda Hu Stephanie I. Mok Anar Shah Erin M. Savner Christos Tolias Roman Corfas Suzhen Chen Perrine Inquimbert Yi Xu Roderick R. McInnes Frank L. Rice Gabriel Corfas Qiufu Ma Clifford J. Woolf Michael E. Greenberg 《Neuron》2010,65(6):886-898
998.
Jung Woong Choi Seyoung Lim Yong-Seok Oh Eung-Kyun Kim Sun-Hee Kim Yun-Hee Kim Kyun Heo Jaeyoon Kim Jung Kuk Kim Yong Ryul Yang Sung Ho Ryu Pann-Ghill Suh 《Cellular signalling》2010,22(7):1153-1161
Among phospholipase C (PLC) isozymes (β, γ, δ, ε, ζ and η), PLC-β plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-β subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-β subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-β1 and PLC-β3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca2+ mobilization were abolished specifically by PLC-β1 silencing, whereas LPA-triggered events were by PLC-β3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-β subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-β1 and BK receptor, while NHERF2 does between PLC-β3 and LPA2 receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-β is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs. 相似文献
999.
Objectives: Translational research using adult stem cells derived from various tissues has been highlighted in cell‐based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta‐derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods: Placental extract, extracted using water‐soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up‐take, PAS (Periodic Acid‐Schiff) staining and urea production were also analysed. Results: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte‐specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human‐based medium, in translational research for regenerative medicine. 相似文献