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941.
Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry 总被引:2,自引:0,他引:2
Hofmann U Seiler M Drescher S Fromm MF 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,766(2):227-233
A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies. 相似文献
942.
Function of dynein and dynactin in herpes simplex virus capsid transport 总被引:24,自引:0,他引:24
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Döhner K Wolfstein A Prank U Echeverri C Dujardin D Vallee R Sodeik B 《Molecular biology of the cell》2002,13(8):2795-2809
After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus. 相似文献
943.
Goti D Balazs Z Panzenboeck U Hrzenjak A Reicher H Wagner E Zechner R Malle E Sattler W 《The Journal of biological chemistry》2002,277(32):28537-28544
During the present study the contribution of lipoprotein lipase (LPL) to low density lipoprotein (LDL) holoparticle and LDL-lipid (alpha-tocopherol (alphaTocH)) turnover in primary porcine brain capillary endothelial cells (BCECs) was investigated. The addition of increasing LPL concentrations to BCECs resulted in up to 11-fold higher LDL holoparticle cell association. LPL contributed to LDL holoparticle turnover, an effect that was substantially increased in response to LDL-receptor up-regulation. The addition of LPL increased selective uptake of LDL-associated alphaTocH in BCECs up to 5-fold. LPL-dependent selective alphaTocH uptake was unaffected by the lipase inhibitor tetrahydrolipstatin but was substantially inhibited in cells where proteoglycan sulfation was inhibited by treatment with NaClO(3). Thus, selective uptake of LDL-associated alphaTocH requires interaction of LPL with heparan-sulfate proteoglycans. Although high level adenoviral overexpression of scavenger receptor BI (SR-BI) in BCECs resulted in a 2-fold increase of selective LDL-alphaTocH uptake, SR-BI did not act in a cooperative manner with LPL. Although the addition of LPL to BCEC Transwell cultures significantly increased LDL holoparticle cell association and selective uptake of LDL-associated alphaTocH, holoparticle transcytosis across this porcine blood-brain barrier (BBB) model was unaffected by the presence of LPL. An important observation during transcytosis experiments was a substantial alphaTocH depletion of LDL particles that were resecreted into the basolateral compartment. The relevance of LPL-dependent alphaTocH uptake across the BBB was confirmed in LPL-deficient mice. The absence of LPL resulted in significantly lower cerebral alphaTocH concentrations than observed in control animals. 相似文献
944.
Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions. 相似文献
945.
946.
Dietzmann J Thiel U Ansorge S Neumann KH Täger M 《Free radical biology & medicine》2002,33(10):1347-1354
The cellular thiol status and its relationship to T-cell activation and cytokine synthesis of mononuclear cells was investigated in patients with end-stage diabetic nephropathy (ESDN) undergoing dialysis treatment. The functional effects of thiol repair by in vitro and in vivo treatment with flavonoids were elucidated. The thiol status of peripheral blood lymphocytes from 30 ESDN patients on hemodialysis and healthy controls was determined by flow cytometry. T-cell activation in response to pokeweed mitogen was analyzed by CD69 expression; cytokines were determined in cell culture supernatants. In result, compared to age-matched healthy subjects, a significant thiol deficiency in ESDN patients was obvious. The lowered total intracellular thiol levels correlated directly to a significant diminished T-cell activation and an elevated synthesis of TNF-alpha in the patient group. The treatment with flavonoids led to a restoration of the thiol status within 72 h in vitro and in vivo. This effect showed a biphasic kinetic that first utilized cell surface thiols and secondly intracellular thiols. In parallel, the T-cell activation was improved substantially along with a significant decrease in TNF-alpha release. These data provide the rational for clinical trials using flavonoids in ESDN to normalize immunoregulatory defects via restoration of the cellular thiol status. 相似文献
947.
Revesz L Di Padova FE Buhl T Feifel R Gram H Hiestand P Manning U Wolf R Zimmerlin AG 《Bioorganic & medicinal chemistry letters》2002,12(16):2109-2112
2,6-Diamino-3,5-difluoropyridinyl substituted pyridinylimidazoles, -pyrroles, -oxazoles, -thiazoles and -triazoles have been identified as novel p38alpha inhibitors. Pyridinylimidazole 11 potently inhibited LPS-induced TNFalpha in mice, showed good efficacy in the established rat adjuvant (ED(50): 10 mg/kg po b.i.d.) and collagen induced arthritis (ED(50): 5 mg/kg po b.i.d.) with disease modifying properties based on histological analysis of the joints. 相似文献
948.
Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling 总被引:1,自引:0,他引:1
Lemmel C Weik S Eberle U Dengjel J Kratt T Becker HD Rammensee HG Stevanovic S 《Nature biotechnology》2004,22(4):450-454
Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands. 相似文献
949.
Mekkriengkrai D Ute K Swiezewska E Chojnacki T Tanaka Y Sakdapipanich JT 《Biomacromolecules》2004,5(5):2013-2019
A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from Hevea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from Hevea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The alpha-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal =CH-CH2OP group. 相似文献
950.
Khaitovich P Weiss G Lachmann M Hellmann I Enard W Muetzel B Wirkner U Ansorge W Pääbo S 《PLoS biology》2004,2(5):E132
Microarray technologies allow the identification of large numbers of expression differences within and between species. Although environmental and physiological stimuli are clearly responsible for changes in the expression levels of many genes, it is not known whether the majority of changes of gene expression fixed during evolution between species and between various tissues within a species are caused by Darwinian selection or by stochastic processes. We find the following: (1) expression differences between species accumulate approximately linearly with time; (2) gene expression variation among individuals within a species correlates positively with expression divergence between species; (3) rates of expression divergence between species do not differ significantly between intact genes and expressed pseudogenes; (4) expression differences between brain regions within a species have accumulated approximately linearly with time since these regions emerged during evolution. These results suggest that the majority of expression differences observed between species are selectively neutral or nearly neutral and likely to be of little or no functional significance. Therefore, the identification of gene expression differences between species fixed by selection should be based on null hypotheses assuming functional neutrality. Furthermore, it may be possible to apply a molecular clock based on expression differences to infer the evolutionary history of tissues. 相似文献