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排序方式: 共有404条查询结果,搜索用时 15 毫秒
1.
RB and the cell cycle: entrance or exit? 总被引:15,自引:0,他引:15
2.
Analysis of liver/bone/kidney alkaline phosphatase mRNA, DNA, and enzymatic activity in cultured skin fibroblasts from 14 unrelated patients with severe hypophosphatasia. 总被引:5,自引:2,他引:3 下载免费PDF全文
M J Weiss K Ray M D Fallon M P Whyte K N Fedde M A Lafferty R A Mulivor H Harris 《American journal of human genetics》1989,44(5):686-694
Hypophosphatasia is a heritable disorder characterized by defective bone mineralization and a deficiency of liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity in serum and tissues. Severe forms of the disease, which are generally lethal in infancy, are inherited in an autosomal recessive fashion. The gene defects that produce hypophosphatasia are poorly understood, but many are likely to occur at the L/B/K ALP locus. To investigate these gene defects, we analyzed L/B/K ALP DNA, RNA, and enzyme activity in cultured dermal fibroblasts from 14 patients with perinatal or infantile hypophosphatasia and from 12 normal individuals. Southern blot analyses of the L/B/K ALP genes from patients and controls revealed identical restriction patterns. Control fibroblast ALP activity correlated with the corresponding L/B/K ALP mRNA levels estimated by blot hybridization analysis and densitometry (r = .94, P less than .0001). In contrast, fibroblasts from the hypophosphatasia patients were deficient in ALP enzyme activity but expressed apparently full-sized L/B/K ALP mRNA at normal levels. Bone specimens from one of the patients were examined and found to be deficient in histochemical ALP but contained immunologic cross-reactive material detected by anti-human liver ALP antiserum. Our results demonstrate that the deficiency of ALP activity in fibroblasts from 14 patients with severe hypophosphatasia is not due to decreased steady-state levels of the corresponding mRNA. The presence of enzymatically inactive L/B/K ALP protein in one of these patients is consistent with a point mutation or small in-frame deletion in the coding region of L/B/K ALP gene. 相似文献
3.
Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH. 相似文献
4.
Summary The distribution of saccharides in pig lymph nodes, particularly on high-endothelial venule (HEV) endothelium and on lymphocytes in these vessels, was studied by examining the binding of fluorescent conjugates of 18 different lectins. Eight of the lectins, particularly with glycan specificity restricted to mannose and polyacetyllactosamine determinants, were found to bind with a high affinity to these structures. Competitive inhibition experiments revealed that polylactosamine-containing glycans were present on endothelia and lymphocytes using lectins from Lycopersicon esculentum and Solanum tuberosum, the latter lectin reacting with lymphocytes only when apparently adherent to the luminal endothelium. The absence on pig endothelium of the Ulex europaeus binding, shown by human endothelia due to the presence of certain fucose epitopes, was confirmed. Pig lymph-node endothelium, however, bound the fucose-specific lectin of Tetragonolobus purpureas, indicating the presence of fucose on pig endothelia in a different conformation to that seen on human endothelia. The results suggested that pig lymph-node HEV endothelium expressed a core fucosylated tri- or tetra-antennary complex glycan with polylactosamine extensions and expressing an Ley determinant.Abbreviations used BS-I
Bandeiraea simplicifolia BS-I
- BS-I-B
B. simplicifolia isolectin B4
- BS-II
B. simplicifolia, lectin II
- FACS
fluorescence-activated cell sorter
- FITC
fluorescein isothiocyanate
- HEV
high-endothelial venule
- LN
lymph node
- MLR
mixed lymphocyte reaction
- PBS
phosphate-buffered saline
- PPME
phosphomannan
- UEA-I
Ulex europeaus lectin I 相似文献
5.
Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities 总被引:2,自引:0,他引:2
The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases. 相似文献
6.
Amphotericin B (AmB) is a membrane-active antibiotic which has been shown to increase ion and small molecule permeability in a variety of model and biological membrane systems. A major mechanistic model, based on BLM systems, proposes that amphotericin forms barrellike pores with cholesterol which are cation selective when added to one side of the membrane and anion selective when added to both sides. We have tested this hypothesis on small and reverse-phase large unilamellar vesicles (SUV and REV) with and without cholesterol. The method used to measure K+, Cl-, and net ion currents is based on ion/H+ exchange detected by the entrapped pH probe pyranine. We find that AmB forms channels which have net selectivity for K+ over Cl- regardless of sidedness or sterol content in SUV. REV with 10% cholesterol also show net K+ selectivity with double-sided addition. Differences are noted between cholesterol- and non-sterol-containing vesicles consistent with at least two separate modes of action: (1) cholesterol-containing SUV form some larger diameter pores which allow the passage of larger ions especially when added to both sides; (2) SUV without sterol form pores which are still K+ over Cl- selective, but larger ions do not pass. The latter mode of action precludes a sterol/pore type of model but not necessarily a barrellike model consisting only of amphotericin molecules.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
8.
Jemima L. Whyte Andrew A. Smith Bo Liu Wilfred R. Manzano Nick D. Evans Girija R. Dhamdhere Mark Y. Fang Howard Y. Chang Anthony E. Oro Jill A. Helms 《PloS one》2013,8(10)
Wnt signaling is required for both the development and homeostasis of the skin, yet its contribution to skin wound repair remains controversial. By employing Axin2LacZ/+ reporter mice we evaluated the spatial and temporal distribution patterns of Wnt responsive cells, and found that the pattern of Wnt responsiveness varies with the hair cycle, and correlates with wound healing potential. Using Axin2LacZ/LacZ mice and an ear wound model, we demonstrate that amplified Wnt signaling leads to improved healing. Utilizing a biochemical approach that mimics the amplified Wnt response of Axin2LacZ/LacZ mice, we show that topical application of liposomal Wnt3a to a non-healing wound enhances endogenous Wnt signaling, and results in better skin wound healing. Given the importance of Wnt signaling in the maintenance and repair of skin, liposomal Wnt3a may have widespread application in clinical practice. 相似文献
9.
10.
Dockrell DH Marriott HM Prince LR Ridger VC Ince PG Hellewell PG Whyte MK 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(10):5380-5388
The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci. 相似文献