Neural differentiation of midbrain cells on various protein-immobilized polyethylene films.

The effect of surface modification of polyethylene (PE) film on differentiation of midbrain (MB) cells obtained from rat embryos was determined by their micromass culture system. When cultured on untreated PE film, cell differentiation was suppressed to approximately two-thirds of that observed in a control culture dish. On the contrary, type I collagen-immobilized PE film increased differentiated foci of the MB cells more than did the untreated PE film. RGDS (Arg-Gly-Asp-Ser) peptide immobilization onto PE film resulted in almost the same differentiation activity as the collagen immobilized PE film. Bovine serum albumin (BSA) immobilization onto PE film enhance the differentiation activity more than did the untreated PE film, but not up to the levels of collagen- and RGDS-immobilized PE. The number of differentiated foci of the MB cells on untreated PE film were increased by the addition of the condition medium prepared from the collagen-immobilized PE film. However, the number of foci was not increased by the addition of other condition media obtained from control dish, untreated, BSA-, and RGDS-immobilized PE. On the other hand, none of these condition media enhanced a differentiation of the neuronal cell line of PC12 cells, suggesting that some factors effectively differentiate midbrain cells, composed of neuronal epithelial and mesenchymal cells, but not the PC12 cells secreted in the condition media prepared from collagen-immobilized PE. In addition, it is probable that neural growth factor was not secreted in these condition media, which could not induce the differentiation of PC12 cells.

Validation of HPLC and GC-MS systems for bisphenol-A leached from hemodialyzers on the basis of FUMI theory.

This paper proposes a method for the validation of chromatography systems in which many experiments to estimate SD or RSD are difficult or impossible to carry out because of time, cost, etc. HPLC systems with UV-Vis and fluorescence detectors and GC-MS system for bisphenol-A leached from hemodialyzers are taken as an example. Examined as validation characteristics are not only the ordinary quantities (precision, accuracy, range, limit of detection (LOD), limit of quantitation (LOQ), specificity and linearity) but also precision plots (measurement RSD vs. concentration), 95% confidence intervals of calibration lines and LOD signals over baselines. The precision plots, calibration confidence intervals and LOD signals are shown to be advantageous to validate and compare the analytical performance of the systems. The LOD, LOQ, precision plots and 95% confidence intervals of calibration lines are all derived from the SD of measurements and the reliability of these quantities and plots depends totally on the reliability of the SD estimates. This paper uses a probability theory, called the FUMI theory, to estimate as exact a measurement SD as possible without the replication. The precision of the HPLC and GC-MS systems is shown to coincide with the repeatability obtained by the repetition of measurements.

Significance of carbohydrate epitopes in a latex allergen with beta-1,3-glucanase activity.

BACKGROUND: One of the latex allergens, Hev b 2, has beta-1,3-glucanase activity. The entire sequence of this allergen is already known. There is one potential N-glycosylation site in this molecule ((27)Asn). Heterogeneous glycosylation of this Asn residue could be a source of the multiplicity of natural Hev b 2. Possible participation of the carbohydrate epitopes of latex beta-1,3-glucanase isoenzymes in their IgE-binding capacity and cross-reactivity was investigated in this study. METHODS: beta-1,3-Glucanase isoenzymes were separated based on their affinities for concanavalin A. IgE-binding capacity and cross-reactivity were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Sequence heterogeneity among the isoenzymes was probed by peptide mass mapping after lysyl endopeptidase digestion. To clarify the relation to Hev b 2, N-terminal sequencing was performed on a fragmented peptide common to the separated isoenzymes. RESULTS: Basic beta-1,3-glucanase was subdivided into two glycosylated isoenzymes (GI and GII) and one non-glycosylated isoenzyme (GIII). IgE antibodies in latex-positive sera chiefly recognized the glycosylated isoenzymes. Inhibition ELISA supported the significance of the carbohydrate epitopes for the IgE recognition and cross-reactivity. However, non-glycosylated GIII, as well as GI and GII, produced positive results in a skin prick test. The three beta-1,3-glucanase isoenzymes shared a partial sequence in common with Hev b 2. CONCLUSIONS: Our results suggest that the carbohydrate epitopes in Hev b 2 homologues are relevant to an in vitro diagnosis of latex allergy and the accompanying cross-reactivity. Carbohydrate epitopes do not necessarily provoke allergic symptoms. Therefore, the actual allergenicity of Hev b 2 and its homologues should be carefully evaluated not only by in vitro IgE tests but also by in vivo tests.