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81.
SM Vlajkovic PR Thorne GD Housley DJ Mu?oz IS Kendrick 《Canadian Metallurgical Quarterly》1998,9(7):1559-1565
There is strong evidence for a purinergic signalling system in the inner ear which regulates auditory sensitivity. This study describes the terminating mechanism for purinergic signalling in the cochlear endolymphatic compartment via ecto-nucleotidases. Exogenous ATP was introduced into the scala media (SM) of the isolated, perfused guinea-pig cochlea, and the effluent was assayed for the adenine nucleotide metabolites by reverse-phase HPLC. Tissue viability was confirmed by fluorescence imaging of cochlear tissues. Extracellular ATP degradation to adenosine was Ca2+/Mg2+ dependent, and was not affected by inhibitors of intracellular ATPases and non-specific alkaline phosphatase. High azide concentration (5 mM) and suramin produced an inhibitory effect on ATP hydrolysis, consistent with inhibition of E-type ATPase activity. The Vmax of ATP hydrolysis (2564 mumol min-1 SM-1) was indicative of high ecto-ATPase activity. Our results support the role of ecto-nucleotidases as a principal mechanism for termination of purinergic signalling within SM, a compartment of the cochlea showing considerable P2X receptor expression. 相似文献
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CD105 (endoglin) is a receptor for transforming growth factor beta (TGFbeta). Although methods to measure soluble forms of TGFbeta and CD105 have been published, no assay is available to quantify the receptor-ligand complexes. We describe both an indirect enzyme-linked immunosorbent assay for the quantitation of soluble CD105-TGFbeta1 and the characterization of the complexes by immunoprecipitation and immunoblotting. Mab E9, specifically reactive with CD105, was utilised as the capture reagent in the ELISA system. Detection of complexes was achieved using chicken antibody against TGFbeta1 and the subsequent detection of bound antibody demonstrated by the addition of anti-species antiserum conjugated to horseradish peroxidase (HRP). By using enhanced chemiluminescence and optimised antibodies, the assay was made sufficiently sensitive and reproducible to detect low levels of circulating complexes. Whether the assay had any practical applications was evaluated in breast cancer patients. Plasma levels of CD105-TGFbeta1 were significantly elevated in 59 patients with breast cancer compared to 52 age matched normal women (p < 0.001). Immunoprecipitation using a rabbit anti-CD105 antibody, which reacts with both dimeric and monomeric CD105, and immunoblotting showed that three molecular forms of CD105-TGFbeta1 complexes > 200, 195, and 125 kDa existed in the plasma. We believe these represent the oligomer, dimer and probably the protease degraded form of CD105 complexed to TGFbeta1. The resistance to hypertonic solution, SDS and heat treatment suggested that the soluble CD105-TGFbeta1 complex may be linked by covalent bonds. The measurement of CD105-TGFbeta complexes in the circulation may have important clinical applications not only in cancer but also in patients with other angiogenic diseases such as rheumatoid arthritis, myocardial infarction and stroke. 相似文献
84.
A Akhmanova FG Voncken H Harhangi KM Hosea GD Vogels JH Hackstein 《Canadian Metallurgical Quarterly》1998,30(5):1017-1027
The anaerobic chytrid Piromyces sp. E2 lacks mitochondria, but contains hydrogen-producing organelles, the hydrogenosomes. We are interested in how the adaptation to anaerobiosis influenced enzyme compartmentalization in this organism. Random sequencing of a cDNA library from Piromyces sp. E2 resulted in the isolation of cDNAs encoding malate dehydrogenase, aconitase and acetohydroxyacid reductoisomerase. Phylogenetic analysis of the deduced amino acid sequences revealed that they are closely related to their mitochondrial homologues from aerobic eukaryotes. However, the deduced sequences lack N-terminal extensions, which function as mitochondrial leader sequences in the corresponding mitochondrial enzymes from aerobic eukaryotes. Subcellular fractionation and enzyme assays confirmed that the corresponding enzymes are located in the cytosol. As anaerobic chytrids evolved from aerobic, mitochondria-bearing ancestors, we suggest that, in the course of the adaptation from an aerobic to an anaerobic lifestyle, mitochondrial enzymes were retargeted to the cytosol with the concomitant loss of their N-terminal leader sequences. 相似文献
85.
GD Stoner C Adams LA Kresty SG Amin D Desai SS Hecht SE Murphy MA Morse 《Canadian Metallurgical Quarterly》1998,19(12):2139-2143
The ability of dietary isothiocyanates to inhibit the esophageal metabolism of N'-nitrosonornicotine (NNN) was examined in F344 rats. Following feeding of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC) or 6-phenylhexyl isothiocyanate for 2 weeks, rats were killed and the esophagi were incubated in vitro with [5-3H]NNN. While dietary BITC, PEITC and PBITC all decreased NNN metabolism, dietary PPITC had the greatest effect, yielding inhibition ranging from 55 to 91% of the control production of various NNN metabolites. To determine the chemopreventive efficacy of PPITC on NNN-induced esophageal tumorigenesis, rats were fed AIN-76A diets containing 0, 1.0 or 2.5 micromol/g PPITC and were given untreated drinking water or drinking water containing 5 p.p.m. NNN. After 87 weeks, the experiment was terminated and the esophageal tumors were counted. Rats that were given untreated drinking water developed no tumors. Rats that were given 5 p.p.m. NNN and unadulterated AIN-76A diet had an esophageal tumor incidence of 71% and a multiplicity of 1.57 tumors/animal. The two dietary concentrations of PPITC reduced the incidence and multiplicity of NNN-induced esophageal tumors by >95%. These results demonstrate the remarkable chemopreventive efficacy of PPITC in the NNN-induced esophageal tumor model. 相似文献
86.
K Reid AM Turnley GD Maxwell Y Kurihara H Kurihara PF Bartlett M Murphy 《Canadian Metallurgical Quarterly》1996,122(12):3911-3919
Melanocytes in the skin are derived from the embryonic neural crest. Recently, mutations in endothelin 3 and the endothelin receptor B genes have been shown to result in gross pigment defects, indicating that this signalling pathway is required for melanocyte development. We have examined the effects of endothelins on melanocyte progenitors in cultures of mouse neural crest. Firstly, they stimulate an increase in progenitor number and act synergistically with another factor, Steel factor, in the survival and proliferation of the progenitors. These findings are consistent with findings from mice with natural mutations in the endothelin receptor B gene, which show an early loss of melanocyte progenitors. Secondly, endothelins induce differentiation of the progenitors into fully mature pigmented melanocytes. This finding is consistent with the expression of endothelins in the skin of mice at the initiation of pigmentation. The melanocytes generated in endothelin-treated cultures also become responsive to alpha melanocyte-stimulating hormone, which then acts to regulate the activity of the pigmentation pathway. These findings indicate two key roles for endothelin in melanocyte development: regulation of expansion of the progenitor pool and differentiation of progenitors into mature melanocytes. 相似文献
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Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL-60 cells were analyzed using 3H-labeled GM3 ([3H]GM3). [3H]GM3 was rapidly internalized into the cells (trypsin-resistant fraction) 8 times more than the control, 3H-labeled GM1 ([3H]GM1). In addition, not only incorporation but also metabolism of [3H]GM3 was more rapid than [3H]GM1 in HL-60 cells. Moreover, one of the metabolites was found to co-migrate with ceramide in thin-layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation-inducible GM3 in HL-60 cells rather than from non-inducing GM1. 相似文献
90.
M González LA Bagatolli I Echabe JL Arrondo CE Argara?a CR Cantor GD Fidelio 《Canadian Metallurgical Quarterly》1997,272(17):11288-11294
The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability. 相似文献