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991.
Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms 总被引:5,自引:2,他引:3
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Adessi C Matton G Ayala G Turcatti G Mermod JJ Mayer P Kawashima E 《Nucleic acids research》2000,28(20):e87
Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies. 相似文献
992.
Tanaka K Uda K Shimada M Takahashi K Gamou S Ellington WR Suzuki T 《Journal of molecular evolution》2007,65(5):616-625
Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role
in cellular energy transactions in animals. Three CK isoform gene families are known—cytoplasmic CK (CK), flagellar CK (fCK),
and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of
other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups
including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted
to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like
ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined
the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes
was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes,
irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon
organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the
view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids. 相似文献
993.
Role of the aminotransferase domain in Bacillus subtilis GabR,a pyridoxal 5′‐phosphate‐dependent transcriptional regulator
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994.
AEF1/MPR25 is implicated in RNA editing of plastid atpF and mitochondrial nad5, and also promotes atpF splicing in Arabidopsis and rice
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995.
996.
Post‐translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris)
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997.
Using a Rebound Matrix to Estimate Consumption Changes from Saving and its Environmental Impact in Japan
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We investigate the extent to which Japanese people can change their consumption and the corresponding environmental impact. We propose a new analytical framework with a rebound matrix that captures the monetary flow from potential savings to their respending (referred to as rebound). A questionnaire is used to derive the matrix. On average, respondents spent 3.4 million Yen annually, resulting in 12.4 tons of carbon dioxide (CO2) emissions in their daily lives. The survey results suggest that acceptable spending reductions would correspond to a CO2 emissions reduction of nearly 6%. However, the CO2 emissions would increase by nearly the same amount when the respondents respend their savable money (rebound CO2 emissions). The annual CO2 emissions and the annually reducible CO2 emissions both increase with the increase in annual expenditure. Consequently, the net CO2 emissions also increase with the increase in annual expenditure. The rebound spending is approximated using the rebound matrix. Finally, it is suggested that the net CO2 emissions can be reduced through lifestyle changes whereby spending on energy items is reduced and the resulting savings are spent on telecommunication, clothes, shoes, education, and housing. 相似文献
998.
Tomohiko Makiyama Hiroyuki Nakamura Nobuo Nagasaka Hisahiro Yamashita Takuya Honda Naoto Yamaguchi Atsushi Nishida Toshihiko Murayama 《Traffic (Copenhagen, Denmark)》2015,16(5):476-492
The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐labeled C6‐ceramide (NBD‐C6‐ceramide, a ceramide having an NBD‐bound C6‐N‐acyl chain) and Golgi‐specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl‐C16‐ceramide‐NBD (a ceramide having an acetylated C‐1 hydroxyl group, C16‐N‐acyl chain, and NBD‐bound C15‐sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl‐C16‐ceramide‐NBD were investigated. Acetyl‐C16‐ceramide‐NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD‐C6‐ceramide, acetyl‐C16‐ceramide‐NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl‐C16‐ceramide‐NBD‐labeled cells before and after mitosis. 相似文献
999.
Xu Lu Dongmei Zhang Hayato Shoji Chengwei Duan Guowei Zhang Tomoya Isaji Yuqin Wang Tomohiko Fukuda Jianguo Gu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(3):598-608
Background
α1,6-Fucosyltransferase-deficient (Fut8?/?) mice displayed increased locomotion and schizophrenia-like behaviors. Since neuroinflammation is a common pathological change in most brain diseases, this study was focused on investigating the effects of Fut8 in microglia and astrocytes.Methods
Brain tissues were analyzed using immunohistochemical staining. Core fucosylation and protein expression were analyzed using lectin blot and western blot, respectively. Fut8-knockout (KO) cells were established by the CRISPR/Cas9 system.Results
The number of Iba-1 positive cells and GFAP positive cells were significantly increased in both untreated and lipopolysaccharide stimulated inflammatory conditional Fut8?/? mice by comparison with both wild-type (Fut8+/+) and hetero (Fut8+/?) mice. Stimulation with pro-inflammatory factors, such as IFN-γ and IL-6, induced expression levels of fucosylation in primary microglia and astrocytes, as well as in glial cell lines. Cell motility and iNOS expression were easily induced by IFN-γ in Fut8-KO BV-2 cells compared with wild-type (WT) cells. In a similar manner, both Fut8-KO C6 cells and primary astrocytes treated with 2-fluoro-L-fucose, a specific inhibitor for fucosylation, showed a higher response to IL-6-stimulated phospho-STAT3 signaling, compared with WT cells.Conclusions
Core fucosylation negatively regulates the states of neuroinflammation by modulating the sensitivity of microglia and astrocytes to inflammatory mediators. The disorders of Fut8?/? mice are caused not only by neurons but also by glial cell dysfunction.General significance
Core fucose is a novel regulator for neuroinflammation in the central nervous system. 相似文献1000.
Mammalian DET1 regulates Cul4A activity and forms stable complexes with E2 ubiquitin-conjugating enzymes 总被引:1,自引:1,他引:0
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Pick E Lau OS Tsuge T Menon S Tong Y Dohmae N Plafker SM Deng XW Wei N 《Molecular and cellular biology》2007,27(13):4708-4719
DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism. 相似文献