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The DNA sequence of the bacteriophage T4 denV gene which encodes the DNA repair enzyme endonuclease V was previously constructed behind the hybrid lambda promoter OLPR in a plasmid vector. The OLPR-denV sequence was subcloned in M13mp18 and used as template to construct site-specific mutations in the denV structural gene in order to investigate structure/function relationships between the primary structure of the protein and its various DNA binding and catalytic activities. The Lys-130 residue of the wild-type endonuclease V has been postulated to be associated with its apurinic endonuclease (AP-endonuclease) activity. The codon for Lys-130 was changed to His-130 or Gly-130, and each denV sequence was subcloned into a pEMBL expression vector. These plasmids were transformed into repair-deficient Escherichia coli (uvrA recA), and the following parameters were examined for cells or cell extracts: expression and accumulation of endonuclease V protein (K-130, H-130, or G-130); survival after UV irradiation; dimer-specific DNA binding; and kinetics of phosphodiester bond scission at pyrimidine dimer sites, dimer-specific N-glycosylase activity, and AP-endonuclease activity. The enzyme's intracellular accumulation was significantly decreased for G-130 and slightly decreased for H-130 despite normal levels of denV-specific mRNA for each mutant. On a molar basis, the endonuclease V gene products generally gave parallel levels of each of the catalytic and binding functions with K-130 greater than H-130 greater than G-130 much greater than control denV-.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Efficient screening of recombinant DNA junctions afforded by probing with synthetic "bridge" oligonucleotides 总被引:1,自引:0,他引:1
Procedures have been developed which simplify and expedite the screening of recombinant DNA constructions for those which only exhibit the desired DNA-DNA junctions. A synthetic DNA oligonucleotide designed to span (or "bridge") sequences around correct restriction enzyme junctions was used as a hybridization probe for the rapid identification of those sequences in several molecular cloning methodologies. It facilitated analyses of the products of random ligation reactions, as well as constructions harbored in bacteria and bacteriophage. "Bridge" probes, [32P]-end-labeled to very high specific activity, remained useful after several hybridizations and often circumvented lengthy restriction analyses. 相似文献
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Lifchez SD Sanger JR Godat DM Recinos RF LoGiudice JA Yan JG 《Plastic and reconstructive surgery》2004,114(5):1068-1076
The ideal donor muscle for facial and hand reanimation has yet to be found. Donor muscles commonly used today, such as the gracilis and pectoralis minor, are limited by bulkiness and the number of force vectors they can provide. In the authors' study of 50 fresh cadaver serratus anterior muscles, they further describe neurovascular anatomy of the muscle slip (i.e., the portion of the muscle that inserts on a rib) and subslip (superficial or deep subdivision of the slip after division along a loose areolar plane). All 260 slips could be separated into a deep and a superficial subslip, yielding a total of 520 subslips. A branch of the serratus artery (a terminal branch of the thoracodorsal artery serving the lower five to seven slips of the muscle) and a branch of the long thoracic nerve were identified for each of these. Deep subslips were thinner than superficial subslips, both at the origin of the slip on the rib periosteum (2.4 mm versus 3.0 mm, p < 0.0001) and centrally at the serratus artery (3.3 mm versus 4.0 mm, p < 0.0001). In addition, the subslips of the most inferior slip were thinner than those of more superior slips, both at the origin of the slip (2.3 mm versus 2.8 mm, p < 0.0001) and at the serratus artery (3.0 mm versus 3.8 mm, p < 0.0001). Fine anastomosing vessels were present between the slips and the subslips. The average number of anastomosing vessels present between adjacent slips was 1.7, and 2.1 anastomosing vessels were present between the subslips of a given slip. Given the thinness of these vessels (all less than 0.2 mm) compared with those of the vascular pedicle of the subslip (mean, 0.7 mm; all greater than 0.4 mm), the authors believe these can be safely divided without compromising subslip vascularity. After division of these vessels, a mean length of 9.6 +/- 1.5 cm is available to allow independent orientation of each subslip. When the serratus muscle flap is separated into its component subslips, a maximum of 10 possible force vectors may be transferred on a single vascular pedicle. Subslips are significantly thinner than donor muscles commonly used today. These two advantages offer the potential for significant functional and aesthetic improvement when the serratus anterior muscle flap is used for face and hand reanimation. Mimetic muscles such as the orbicularis oculi and orbicularis oris could possibly be reconstructed in their proper anatomical positions. 相似文献
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WW or WoW: the WW domains in a union of bliss 总被引:1,自引:0,他引:1
WW domains are small protein modules that recognize proline-rich peptide motifs or phosphorylated-serine/threonine proline sites in cognate proteins. Within host proteins these modules are joined to other protein domains or to a variety of catalytic domains acting together as adaptors or targeting anchors of enzymes. An important aspect of signaling by WW domains is their ability to recognize their cognate ligands in tandem. Tandem WW domains not only act in a synergistic manner but also appear to chaperone the function of each other. In this review, we focus on structure, function, and mechanism of the tandem WW domains co-operativity as well as independent actions. We emphasize here the implications of tandem arrangement and cooperative function of the domains for signaling pathways. 相似文献
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Histopathological effects of cisplatin,doxorubicin and 5-flurouracil (5-FU) on the liver of male albino rats
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Hassan I El-Sayyad Mohamed F Ismail F M Shalaby RF Abou-El-Magd Rajiv L Gaur Augusta Fernando Madhwa HG Raj Allal Ouhtit 《International journal of biological sciences》2009,5(5):466-473
Cisplatin, doxorubicin and fluorouracil (5-FU), drugs belonging to different chemical classes, have been extensively used for chemotherapy of various cancers. Despite extensive investigations into their hepatotoxicity, there is very limited information on their effects on the structure and ultra-structure of liver cells in vivo. Here, we demonstrate for the first time, the effects of these three anticancer drugs on rat liver toxicity using both light and electron microscopy. Light microscopic observations revealed that higher doses of cisplatin and doxorubicin caused massive hepatotoxicity compared to 5-FU treatment, including dissolution of hepatic cords, focal inflammation and necrotic tissues. Interestingly, low doses also exhibited abnormal changes, including periportal fibrosis, degeneration of hepatic cords and increased apoptosis. These changes were confirmed at ultrastructural level, including vesiculated rough endoplasmic reticulum and atrophied mitochondria with ill-differentiated cisternae, dense collection of macrophages and lymphocytes as well as fibrocytes with collagenous fibrils manifesting early sign of fibrosis, especially in response to cisplatin and doxorubicin -treatment. Our results provide in vivo evidence, at ultrastructural level, of direct hepatotoxicity caused by cisplatin, doxorubicin and 5-FU at both light and electron microscopi. These results can guide the design of appropriate treatment regimen to reduce the hepatotoxic effects of these anticancer drugs. 相似文献
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Joana RF Abreu Daphne de Launay Marjolein E Sanders Aleksander M Grabiec G Marleen van de Sande Paul P Tak Kris A Reedquist 《Arthritis research & therapy》2009,11(4):R121-13
Introduction
Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS. 相似文献9.
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Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals. 相似文献