BioZyme (United States)

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

A study on how the covalent character of the halogen bond can be modulated by using different halogen donors.

The objective was to determine if differences in days in milk (DIM) affect apparent nutrient digestibility in lactating multiparous cows fed the same ration. Of 136 cows in a multiparous, high-producing pen on a 1,000-cow commercial dairy, 24 were selected for sampling based on differences in DIM and milk yield (Low DIM: n = 12, milk = 64 ± 6 kg, and DIM = 85 ± 6; High DIM: n = 12, milk = 55 ± 4 kg, and DIM = 158 ± 15). Milk, fecal, and TMR samples were collected once daily for 10 consecutive days. Samples were collected in the morning on 6 d and the afternoon on 4 d. Individual cow milk and fecal samples were composited into one daily milk or fecal sample per DIM group and analyzed for nutrient composition. In addition, daily fecal composite and TMR samples were analyzed for apparent digestibility of potentially digestible NDF (PdNDFd), NDF (NDFd), protein (Protd), and starch (Starchd). Fecal nutrient composition and apparent digestibility data were analyzed using the Glimmix procedure of SAS. The model included fixed effects of group, sampling time, and their interaction. Day was included as a repeated measure. The CORR and REG procedures were used for comparing daily variations in digestibility with milk yield and composition. For fecal nutrient composition, DIM × sampling time was significant for ADF and NDF, due to sampling time differences in the low-DIM group (Table 1). There was a group effect on fecal DM in which DM was lower in low-DIM cows. Time effects were also observed, with CP being higher before noon and ADF, NDF, and ash being higher after noon. There was no effect of DIM on PdNDFd, NDFd, Protd, or Starchd. There was an effect of DIM × sampling time on Starchd (P = 0.03) due to higher Starchd at the before-noon sampling for high-DIM cows (P = 0.03). There were no significant correlations of digestibility measures with milk yield. A 1% increase in PdNDFd (P = 0.02) and NDFd (P = 0.006) was associated with a 0.04 and 0.05% increase, respectively, in milk fat percentage. A 1% increase in Starchd (P = 0.003) was associated with a 0.03% increase in milk protein percentage. Because DIM had minimal effects on digestibility measures, it is likely not necessary to consider DIM when selecting cows for estimation of nutrient digestibility within a high-producing pen.

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.

The low affinity IgE receptor, FcepsilonRII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity of ADAM (a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.