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排序方式: 共有233条查询结果,搜索用时 421 毫秒
1.
Quantitation of free sphingosine in liver by high-performance liquid chromatography 总被引:10,自引:0,他引:10
A H Merrill E Wang R E Mullins W C Jamison S Nimkar D C Liotta 《Analytical biochemistry》1988,171(2):373-381
Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt. 相似文献
2.
NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice. 总被引:20,自引:4,他引:16
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U P Thorgeirsson T Turpeenniemi-Hujanen J E Williams E H Westin C A Heilman J E Talmadge L A Liotta 《Molecular and cellular biology》1985,5(1):259-262
NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing. 相似文献
3.
Purification and characterization of a murine basement membrane collagen-degrading enzyme secreted by metastatic tumor cells 总被引:13,自引:0,他引:13
A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and type IV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m. The apparent molecular weight of the enzyme was 160,000 but about 70,000 when Triton X-100 was added to the column buffer. The purified enzyme protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptide chains of about 68,000 and 62,000 daltons. The enzyme activity could be increased by preincubation with trypsin and it is possible that the two chains represent latent and active enzyme forms. The enzyme activity was not reduced in the presence of dithiothreitol, it had a pH optimum of 7.6 and was inhibited by EDTA but not N-ethylmaleimide, phenylmethylsulfonyl fluoride, or Trasylol. The inhibition with EDTA was reversible. The pro-alpha 1(IV) and pro-alpha 2(IV) chains of the type IV procollagen substrate were both degraded at a similar rate to form two pairs of degradation fragments corresponding in molecular weights to about 70 and 30% of the original size chains. The presence of Triton X-100 increased slightly the activity of the enzyme and diminished the reduction of its activity upon freezing, indicating that the enzyme is a hydrophobic protein. 相似文献
4.
The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor 总被引:10,自引:0,他引:10
R P Mecham A Hinek G L Griffin R M Senior L A Liotta 《The Journal of biological chemistry》1989,264(28):16652-16657
Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2058 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin-binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor. 相似文献
5.
C W Slife E Wang R Hunter S Wang C Burgess D C Liotta A H Merrill 《The Journal of biological chemistry》1989,264(18):10371-10377
Long-chain (sphingoid) bases may serve as another category of "lipid second messenger" because they inhibit protein kinase C and affect multiple cellular functions. Free sphingosine has been found in rat liver (Merrill, A. H., Jr., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D. C. (1988) Anal. Biochem. 171, 373-381); hence, this study determined if liver plasma membranes contain free long-chain bases and have the ability to form them from endogenous enzymes and substrates. Isolated plasma membranes contained 0.45 nmol of sphingosine/mg of protein which, based on the recovery of the membranes, was equivalent to 3.5 +/- 1.2 nmol/g of liver and at least half of the total free sphingosine in liver. When the membranes were incubated at 37 degrees C, the amount increased at an initial rate of 5-25 pmol/min/mg, resulting in a 2-3-fold increase over an hour. Sphingosine formation required divalent cations, was optimal at neutral to alkaline pH, and was temperature-dependent. Activities with these characteristics were not identified in microsomes or lysosomes (lysosomal activities with acidic pH optima were detected, however); hence, they appear to reflect a separate plasma membrane system. Sphingosine formation was stimulated by ceramides either added exogenously or formed endogenously by treating the membranes with sphingomyelinase (but not endoglycoceramidase). Sphingomyelin hydrolysis to ceramide was also observed during incubation of the plasma membranes alone. Some of the properties of this system resembled the neutral sphingomyelinase and ceramidase activities of liver. While the physiological significance of this endogenous sphingosine is not known, this system has the appropriate subcellular location to provide sphingosine as a participant in signal transduction. 相似文献
6.
Comparative metabolic clearance rates of beta-endorphin and beta-lipotropin in humans 总被引:1,自引:0,他引:1
In normal human subjects under basal conditions, we have reported that molar concentrations of immunoreactive β-lipotropin (IR-β-LPH) are approximately threefold greater than those of IR-β-endorphin (β-Ep). Following acute stimulation, there is a further two- to threefold disproportionate rise in plasma concentrations of IR-β-LPH as compared to those of IR-β-Ep. To begin to assess the possible factors involved in such altered IR-β-LPH/IR-β-Ep ratios in plasma, the metabolic clearance rate (MCR), volume of distribution (Vd), fractional rate of disappearance (Kd), and half-life () of these peptides were determined by means of bolus injection of highly purified human β-LPH and synthetic human β-Ep in normal human subjects. β-Ep was found to have an MCR and a Kd greater than that of β-LPH, and a shorter . These differences, however, although they may in part be contributory, cannot solely account for the greater ratio of IR-β-LPH to IR-β-Ep in plasma, or for the disproportionate rise in plasma concentrations of these peptides after acute stimulation. 相似文献
7.
Marina Giuliano Mauro Andreotti Giuseppe Liotta Haswell Jere Jean-Baptiste Sagno Martin Maulidi Sandro Mancinelli Ersilia Buonomo Paola Scarcella Maria F. Pirillo Roberta Amici Susanna Ceffa Stefano Vella Leonardo Palombi Maria Cristina Marazzi 《PloS one》2013,8(7)
Background
Optimized preventive strategies are needed to reach the objective of eliminating pediatric AIDS. This study aimed to define the determinants of residual HIV transmission in the context of maternal antiretroviral therapy (ART) administration to pregnant women, to assess infant safety of this strategy, and to evaluate its impact on maternal disease.Methodology/Principal Findings
A total of 311 HIV-infected pregnant women were enrolled in Malawi in an observational study and received a nevirapine-based regimen from week 25 of gestation until 6 months after delivery (end of breastfeeding period) if their CD4+ count was > 350/mm3 at baseline (n = 147), or indefinitely if they met the criteria for treatment (n. 164). Mother/child pairs were followed until 2 years after delivery. The Kaplan-Meier method was used to estimate HIV transmission, maternal disease progression, and survival at 24 months. The rate of HIV infant infection was 3.2% [95% confidence intervals (CI) 1.0-5.4]. Six of the 8 transmissions occurred among mothers with baseline CD4+ count > 350/mm3. HIV-free survival of children was 85.8% (95% CI 81.4-90.1). Children born to mothers with baseline CD4+ count < 350/mm3 were at increased risk of death (hazard ratio 2.6, 95% CI 1.1-6.1). Among women who had stopped treatment the risk of progression to CD4+ count < 350/mm3 was 20.6% (95% CI 9.2-31.9) by 18 months of drug discontinuation.Conclusions
HIV transmission in this cohort was rare however, it occurred in a significative proportion among women with high CD4+ counts. Strategies to improve treatment adherence should be implemented to further reduce HIV transmission. Mortality in the uninfected exposed children was the major determinant of HIV-free survival and was associated to maternal disease stage. Given the considerable proportion of women reaching the criteria for treatment within 18 months of drug discontinuation, life-long ART administration to HIV-infected women should be considered. 相似文献8.
Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity-based processes. Serum proteomic pattern diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. This approach has recently shown tremendous promise in the detection of early-stage cancers. The biomarkers found by SELDI-TOF-based pattern recognition analysis are mostly low molecular weight fragments produced at the specific tumor microenvironment. 相似文献
9.
Krieg RC Liotta LA Petricoin EF Herrmann PC 《Journal of biochemical and biophysical methods》2004,58(2):119-124
Measurement of carbon dioxide levels has been employed to follow cellular metabolic reactions for quite some time. By radio-labeling substrate molecules and evaluating the radioactivity levels of the carbon dioxide released, insight into metabolic pathways can be gleaned. Currently, no carbon dioxide capturing device is available that can be used with large volume cell monolayers growing under standard conditions within a regular commercially available culture flask. In this note we describe a simple device for collecting radio-labeled carbon dioxide from a standard culture flask. The device is independent of the culture flask, but can be attached for metabolic measurements allowing cells to be grown under standard conditions prior to study. The presented design permits convenient transfer of the device between flasks without contaminating or disturbing cells growing within the flasks. Data are presented demonstrating the reproducibility of measurements made with multiple devices with different substrate concentrations and varying periods of time, ranging up to 3 h. 相似文献
10.
Mao S Bouygues M Welch C Biba M Chilenski J Schinazi RF Liotta DC 《Bioorganic & medicinal chemistry letters》2004,14(19):4991-4994
The beta-D-enantiomer of FDOC (2',3'-dideoxy-5-fluoro-oxacytidine) exhibits potent anti-HIV-1 activity. It was obtained in optically pure form by employing a tandem kinetic resolution/chiral salt crystallization protocol. In addition, conditions were developed that allowed the unwanted butyrate ester of the L-enantiomer of FDOC to be racemized. This material could then be recycled in future resolutions. 相似文献