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A method for the accurate determination of the melting temperature (Tm) of surface-immobilized DNA duplexes that exploits the fluorescence-quenching properties of gold is reported. A thiolated single-stranded DNA probe is chemisorbed onto a gold surface and then hybridized to a fluorophore-labeled complementary sequence. On formation of the duplex, the fluorescence of the label is effectively quenched by the gold surface. As the temperature is increased and the duplex denatures, the fluorophore label moves away from the gold surface and the fluorescence signal is again observed. The increase in fluorescence is measured as the temperature is ramped, and using first-derivative plots, the Tm is determined. To demonstrate the approach, the Tm of the cystic fibrosis DF508 mutation was determined in three different phases: in solution, in suspension immobilized on gold nanoparticles, and immobilized on gold film-coated substrate. The technique was further applied to optimize conditions for differentiation between a surface-immobilized DF508 mutant probe and a mutant/wild-type target exploiting increasing stringency in varying salt and formamide concentrations. The approach has application in optimization of assay conditions for biosensors that use gold substrates as well as in melting curve analysis. 相似文献
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Ryuichi Suwa Hiroaki Hakata Hiromichi Hara Hany A. El-Shemy Joseph J. Adu-Gyamfi Nguyen Tran Nguyen Synsuke Kanai David A. Lightfoot Pravat K. Mohapatra Kounosuke Fujita 《Plant Physiology and Biochemistry》2010,48(2-3):124-130
Short hot and dry spells before, or during, silking have an inordinately large effect on maize (Zea mays L.; corn) grain yield. New high yielding genotypes could be developed if the mechanism of yield loss were more fully understood and new assays developed. The aim here was to determine the effects of high temperature (35/27 °C) compared to cooler (25/18 °C) temperatures (day/night). Stress was applied for a 14 d-period during reproductive stages prior to silking. Effects on whole plant biomass, ear development, photosynthesis and carbohydrate metabolism were measured in both dent and sweet corn genotypes. Results showed that the whole plant biomass was increased by the high temperature. However, the response varied among plant parts; in leaves and culms weights were slightly increased or stable; cob weights decreased; and other ear parts of dent corn also decreased by high temperature. Photosynthetic activity was not affected by the treatments. The 13C export rate from an ear leaf was decreased by the high temperature treatment. The amount of 13C partitioning to the ears decreased more than to other plant parts by the high temperature. Within the ear decreases were greatest in the cob than the shank within an ear. Sugar concentrations in both hemicellulose and cellulose fractions of cobs in sweet corn were decreased by high temperature, and the hemicellulose fraction in the shank also decreased. In dent corn there was no reduction of sugar concentration except in the in cellulose fraction, suggesting that synthesis of cell-wall components is impaired by high temperatures. The high temperature treatment promoted the growth of vegetative plant parts but reduced ear expansion, particularly suppression of cob extensibility by impairing hemicellulose and cellulose synthesis through reduction of photosynthate supply. Therefore, plant biomass production was enhanced and grain yield reduced by the high temperature treatment due to effects on sink activity rather than source activity. Heat resistant ear development can be targeted for genetic improvement 相似文献
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Jér?me Verine Jacqueline Lehmann-Che Hany Soliman Jean-Paul Feugeas Jean-Sébastien Vidal Pierre Mongiat-Artus Stéphanie Belhadj Josette Philippe Matthieu Lesage Evelyne Wittmer Stéphane Chanel Anne Couvelard Sophie Ferlicot Nathalie Rioux-Leclercq Jean-Michel Vignaud Anne Janin Stéphane Germain 《PloS one》2010,5(4)
Background
We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors.Methodology/Principal Findings
Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status.Conclusions/Significance
Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10−49, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction. 相似文献65.
Canavan disease is a genetic disorder associated with optic neuropathy and the metabolism of N-acetylaspartate is defective in this disorder due to mutations in the gene coding for the enzyme aspartoacylase II. Here we show that the plasma membrane transporter NaDC3, a Na+-coupled transporter for dicarboxylates, is able to transport N-acetylaspartate, suggesting that the transporter may function in concert with aspartoacylase II in the metabolism of N-acetylaspartate. Since Canavan disease is associated with ocular complications, we investigated the expression pattern of NaDC3 and aspartoacylase II in ocular tissues in mouse by in situ hybridization. These studies show that NaDC3 mRNA is expressed in the optic nerve, most layers of the retina, retinal pigment epithelium, ciliary body, iris, and lens. Aspartoacylase II mRNA is coexpressed in most of these cell types. We conclude that transport of N-acetylaspartate into ocular tissues via NaDC3 and its subsequent hydrolysis by aspartoacylase II play an essential role in the maintenance of visual function. 相似文献
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Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding 总被引:1,自引:0,他引:1
Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes. 相似文献
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El-Shemy HA Aboul-Enein AM Aboul-Enein MI Issa SI Fujita K 《Journal of biochemistry and molecular biology》2003,36(4):387-389
The young developing leaves of willow (Salix safsaf, Salicaceae) trees have antileukemic activity. After a 24-h incubation in vitro, the crude water extracts of the leaves killed a majority of the blasts of acute myeloid leukemia (AML, 73.8%). 相似文献
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Farwanah H Neubert R Zellmer S Raith K 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,780(2):443-450
The separation of the major stratum corneum lipids, i.e., ceramides, fatty acids, cholesterol and its esters by means of high-performance thin-layer chromatography is hereby presented. The used automated multiple development technique allows the reproducible development of a 17-step solvent gradient also capable of separating seven ceramide classes in the same run. Reliable quantification has been performed after visualisation and densitometric scanning. The present approach is less time and solvent-consuming than previously described procedures. The application to samples obtained by in vivo skin surface extraction with hexane-ethanol (2:1) demonstrates that the method can be routinely used for diagnostic purposes. 相似文献
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Elsheikha HM Mackenzie CD Rosenthal BM Marteniuk JV Steficek B Windsor S Saeed AM Mansfield LS 《The Journal of parasitology》2005,91(4):877-881
Fourteen miniature donkeys (Equus asinus) in a mid-Michigan herd of 38 animals presented with clinical signs of besnoitiosis, including the presence of typical tissue cysts in the ocular sclera, the buccal and nasal mucosa, together with characteristic dermatitis in specific areas of the body. The common histopathological change seen was the presence of many 100-200-microm diameter, thick walled, typical Besnoitia sp. tissue cysts together with a chronic cellular response associated with degenerating cysts. Microscopy of isolated scleral cysts and skin biopsies showed the presence of protozoal organisms consistent in morphology with that of Besnoitia bennetti bradyzoites. Molecular analysis of these parasites indicates that they differ from previously described coccidia, including Besnoitia sp., from rabbits and opossums. Isolated cases of infection with this agent have been reported infrequently in equids; however, this is the first report of an outbreak in a herd of donkeys in the United States. 相似文献