PubMed · 4957754
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L F Craver, D G Miller. Multiple myeloma.. https://doi.org/10.3322/canjclin.16.4.142
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BACKGROUND: Analytical evaluations of an available system for capillary zone electrophoresis (CZE) of serum protein have been reported. However, data concerning long-term precision and stability of the system, operated under routine conditions, are lacking. We report data from an internal quality control (QC) scheme, obtained over a 1-year period. METHODS: Measurements were done with a pair of instruments (Beckman Paragon CZE 2000 system), each equipped with seven capillaries. After preliminary (1 month) assessment of possible inter-capillary and inter-instrument variations, the QC material (a home prepared serum pool stored in the frozen state) was assayed daily over a 1-year period. RESULTS: Maximum inter-capillary and inter-instrument differences were 3.1% and 2.4%. No significant trend was observed for daily values (205 measurements over 1 year); in the same period overall imprecision values (CV) were in the interval 1.2% (albumin) to 3.2-6.1% (globulin zones). Mean monthly imprecision (CV) values were in the interval 1.1% (albumin) to 5.2% (globulin zones). There was no significant trend of monthly means with time. The observed imprecision values were within the biological variation-derived goals for imprecision. CONCLUSIONS: It is concluded that the assessed analytical instruments, operated in routine conditions, show long-term stability and imprecision consistent with the clinical use of the results produced.
Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22(phox) as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.
Viral respiratory infections rapidly increase vascular permeability, which leads to the transudation of serum proteins into airway secretions and tissues. To determine whether this process activates airway epithelial cells, bronchial epithelial cells were incubated with serum, and interleukin (IL)-8 secretion and gene expression were examined. As little as 0.1% serum significantly enhanced IL-8 secretion, and maximal secretion (65 +/- 4 ng/ml, 48 h) was observed with 10% serum. Low-density lipoprotein, but not albumin or immunoglobulin G, augmented bronchial epithelial IL-8 secretion, which was partially blocked by a monoclonal antibody specific for the low-density lipoprotein receptor. The IL-8-inducing activity of plasma was also augmented by clotting and platelet activation. Mechanistically, serum activated nuclear factor-kappaB and increased the stability and steady state levels of IL-8 mRNA. In summary, specific components of serum are potent activators of IL-8 mRNA and secretion, and the increased IL-8 production is likely to be a result of both increased transcription and mRNA stability. This effect may represent an innate mechanism for the recruitment of neutrophils to the airway in response to noxious stimuli, such as viral infections, that increase vascular permeability.