On the pinning of interfaces on micropillar edges.

Microsystems for biotechnology often make use of pillars to perform the targeted microfluidic functions. In many cases the role of the pillars is to block or maintain fixed an interface between two immiscible fluids. This phenomenon is usually called pinning. The pining principle is used for capillary valves, liquid-liquid extraction devices, etc. It is common to estimate the pinning efficiency by considering mathematically perfect edges. In reality, microfabricated edges always show some smoothness that can be modeled by a small curvature radius. In this work, we investigate the pinning on square, circular, triangular, and diamond-shaped pillars, and analyze the anchoring on the upstream edges (first pinning conditions) and possibly on the downstream edges (second pinning conditions). It is shown that pinning efficiency decreases very quickly with the curvature radius of the pillar edge. It is concluded that the quality of the microfabrication is essential. Especially oxidation of the silicon reduces considerably the pinning efficiency. Moreover, it is shown that square pillars pin better an interface than triangular pillars. For triangular pillars, a pillar angle--angle between two facets--optimal for pinning has been determined that depends on the quality of microfabrication.

A concatenated form of Epstein-Barr viral DNA in lymphoblastoid cell lines induced by transfection with BZLF1.

The replicative form of Epstein-Barr virus (EBV) DNA was studied using two lymphoblastoid cell lines, X50-7 and 6F11, which are latently infected by Epstein-Barr virus. The lytic cycle of EBV infection was induced by transfection of the cells with the BRLF1/BZLF1 coding region of the P3HR-1 defective genome. We combined two techniques to identify the productive replicative form of Epstein-Barr viral DNA in the lytic cycle-induced cells. Restriction enzyme analysis followed by Southern blot hybridization identified a significant increase in the fused fragment encompassing both ends of EBV DNA. This indicates an increase in either episomal DNA or concatameric linear DNA. Southern blot analysis of in situ lysing gels revealed that the cellular content of linear EBV DNA was also increased significantly after the initiation of the viral lytic cycle, while the amount of circular DNA remained approximately constant. We propose from these results that the source of the fused fragment encompassing both ends of EBV DNA is a concatenated linear EBV DNA molecule, and that such a concatenated molecule most likely represents a replicative form of EBV DNA in productively infected cells.

Kinetics of reduction by substrate or dithionite and heme-heme electron transfer in the multiheme hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase of Nitrosomonas catalyzes the dehydrogenation of NH2OH. It contains hemes c553, c559 and P460 in the ratio 5:2:1. At equilibrium four or five c hemes are reduced by NH2OH or NH2NH2, respectively. Heme P460 is the site of electron entry into the enzyme; electrons exit via P460 to O2 or H2O2 with rate constants of 30s-1. We report that hydroxylamine oxidoreductase has two categories of electron-accepting sites: (a) heme P460, an H2O2-sensitive site, which is reactive with NH2OH (2.2 hemes c557 and 2 hemes c559 are reduced) or NH2NH2 (3.3 heme c 553 and 2 heme c559 are reduced) and (b) an H2O2-insensitive site(s) which is reactive with H2O2 (approximately 0.15 heme c553 is reduced); hydroquinone, pyrogallol, N-methyl hydroxylamine, pyocyanine, and ascorbate (approximately 0.8 heme c553 is reduced); or Na2S2O4 or EDTA-photoreduction with proflavin, deazalumiflavin or acridine orange and methylviologen (all hemes are reduced). The rate constants at 19 degrees C for reduction by dithionite were: 0.7 heme c553 (7s-1), 4.3 hemes c553 (0.07 s-1), 0.7 heme c559 (0.8s-1), 1.3 hemes c559 (0.1s-1), P460 (0.013s-1). At 2 degrees C the rate constant for 0.8 heme c559 was 1.7s-1. The data indicate that one heme c552 is reduced by dithionite at the same rate as mammalian cytochrome c; other hemes are reduced much more slowly and are possibly inaccessible to the solvent. The rate constants at 2 degrees C for reduction by NH2OH were: 1.8 hemes c553 (30s-1), 0.2 heme c553 (2.4s-1), 1.7 hemes c559 (19s-1), 0.3 heme c559 (1.4s-1). For reduction by NH2NH2 the values were: 2.6 hemes c553 (23s-1), 0.7 heme c553 (1.6s-1), 1.3 hemes c559 (22s-1), 0.7 heme c559 (4.2s-1). Thus reduction by NH2OH at the substrate site was at least an order of magnitude faster than reduction of hydroxylamine oxidoreductase heme by Na2S2O4. Comparison of rates of heme-heme electron transfer on the enzyme during reoxidation by O2 or H2O2, reduction by Na2S2O4 and reduction by NH2OH or NH2NH2 indicates that the enzyme can exist in distinct states which result in different rates of heme-heme electron transfer. Comparison of the rate of substrate reduction of c hemes of hydroxylamine oxidoreductase (HAO) with the turnover of the enzyme in vivo is consistent with the electron path NH2OH----HAO P460----HAO c hemes----biological electron acceptor.