Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.

We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.

Polycarbonate-urethane hard segment type influences esterase substrate specificity for human-macrophage-mediated biodegradation.

Previous studies have shown that esterase activity can degrade a variety of polyurethanes (PUs), including polycarbonate-based PUs (PCNUs). When cultured on PCNUs, differing in their chemistries, monocyte-derived macrophages (MDM) synthesized and secreted different amounts of both cholesterol esterase (CE) and monocyte-specific esterase (MSE). MDM were seeded on PCNUs synthesized with hexane diisocyanate (HDI) or 4,4'-methylene-bis-phenyl diisocyanate (MDI), PCN and [14C]butanediol (BD) in the ratio 3:2:1 (referred to as HDI321 or MDI321). The effect of phenylmethylsulfonyl fluoride (PMSF, a serine esterase and proteinase inhibitor), sodium fluoride (NaF, a MSE inhibitor) and sodium taurocholate (NaT, a CE stimulator) was assessed on degradation (measured by radiolabel release (RR)) and esterase activity in MDM lysate. The results were compared to the effect that these reagents had on commercially available CE and carboxyl esterase (CXE), which has a specificity similar to MSE. NaF inhibited CXE- and MDM-mediated RR to the same extent as for both PCNUs. However, the MDM-mediated RR from MDI321 was 1.8-times higher than HDI321 in the presence of NaT (P = 0.005). This study suggests that the difference in diisocyanate chemistry may dictate the relative contribution of each esterase to a specific material's degradation. This may be related to both the substrate specificity of each esterase, as well as by the relative amount of each esterase that the specific biomaterial substrates induce the cells to synthesize and secrete.

The human macrophage response during differentiation and biodegradation on polycarbonate-based polyurethanes: dependence on hard segment chemistry.

Human monocytes, isolated from whole blood, were seeded onto tissue culture grade polystyrene (PS) and three polycarbonate-based polyurethanes (PCNUs) (synthesized with either 1,6-hexane diisocyanate (HDI) or 4,4'-methylene bis-phenyl diisocyanate (MDI), poly(1,6-hexyl 1,2-ethyl carbonate) diol (PCN) and 1,4-butanediol (BD) in different stoichiometric ratios (HDI:PCN:BD 4:3:1 or 3:2:1 and MDI:PCN:BD 3:2:1) (referred to as HDI431, HDI321 and MDI321, respectively). Following their differentiation to monocyte-derived macrophages (MDMs) the cells were trypsinized and reseeded onto each of the PCNUs synthesized with either 14C-HDI or 14C-BD and degradation was measured by radiolabel release (RR). When the differentiation surface was MDI321, there was more RR from 14C-HDI431 than from any other surface (p < 0.0001) whereas the amount of esterase (identified by immunoblotting) as well as the esterase activity was the greatest in MDM differentiated on PS, reseeded on 14C-HDI431 (p < 0.0001). The effect of potential degradation products (methylene dianiline (MDA) and BD) from the PCNUs was carried out to determine possible links between products and substrate-induced activation of MDM. MDA was found to inhibit RR 60% from MDM seeded on 14C-MDI321B (p < 0.0001), approximately 20% from 14C-HDI431 (p = 0.002) and no effect from 14C-HDI321B. MDA inhibited esterase activity 30% from MDM only on 14C-MDI321B (p = 0.003), but no effect on esterase activity was observed for the other two polymers. BD had no inhibitory effect on RR from any PCNU, but did inhibit esterase activity in MDM on 14C-HDI431 (p = 0.025). This study indicates that the degradation of a specific material is a multi-factorial process, dictated by its susceptibility to hydrolysis, the effect of specific products generated during this course of action, and perhaps not as well appreciated, the material's inherent ability to influence enzyme synthesis and release.