Microfluidics is poised to have an impact on life sciences research. However, current microfluidic methods are not compatible with existing laboratory liquid dispensing and detection infrastructure. This incompatibility is a barrier to adoption of microfluidic systems and calls for improved approaches that will enhance performance and promote acceptance of microfluidic systems in the life sciences. Ease of use, standardized interfaces and automation remain critical challenges. We present a platform based on surface tension effects, where the difference in pressure inside drops of unequal volume drives flow in passive structures. We show integration with existing laboratory infrastructure, microfluidic operations such as pumping, routing and compartmentalization without discrete micro-components as well as cell patterning in both monolayer and three-dimensional cell culture.

The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.The human UV-damaged-DNA binding protein has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E). Cells from about 30% of XP-E patients (6 of 19) were shown to lack the damaged-DNA binding activity of DDB (3,27). DDB was originally identified as an activity that binds to UV-damaged DNA (3). It has high affinities for the 6-4 photoproduct (14,29,36). In addition, DDB also binds to cisplatin-modified DNA (14). It has been proposed that the damaged-DNA binding activity of DDB is related to a potential DNA repair function (7,15,18,20,36). The microinjection of purified DDB overcomes the repair deficiencies in cells from XP-E patients lacking the damaged-DNA binding activity of DDB (20, 27). However, purified DDB has no significant effect in nucleotide excision repair assays in vitro (18). A recent study proposed that DDB functions as a repair protein in the context of chromatin structure (27). A repair function of DDB would be consistent with the fact that the p48 subunit of DDB possesses extensive sequence homology with the Cockayne syndrome protein CS-A, which is involved in transcription-coupled DNA repair (2,9,12,19).DDB also possesses a transcriptional functi...

The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, we looked for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. Here we show that DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. We show that the UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. Moreover, the 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1 depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts resulted in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also revealed a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, was enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. We also observed evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulated E2F1-activated transcription. In addition, the transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 was stimulated by coexpression of DDB. Moreover, DDB expression could overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. We speculate that the damaged-DNA binding function of DDB, besides repair, might serve as a negative regulator of E2F1-activated transcription, as damaged DNA will sequester DDB and make it unavailable for E2F1. Furthermore, the binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA.E2F1 is the most-studied member of the E2F family of transcription factors. E2F1 binds the consensus E2F site (TT TCGCGC) as a heterodimer in conjunction with DP1, and it stimulates transcription both in vitro and in vivo (3,13,(20)(21)(22)31). Several genes that are essential for DNA replication and S-phase entry have been shown to be transcriptionally activated by overexpression of E2F1. Included are genes expressing dihydrofolate reductase (4, 26, 56), ribonucleotide reductase, PCNA, DNA polymerase ␣, thymidine kinase, cyclin E, cyclin A, and E2F1, as well as cdc2 (8,42,44). This is consistent with the observation that overexpression of E2F1 induces quiescent cells to enter S phase (2, 30).The activity and levels of E2F1 are regulated very tightly during the progression of the cell cycle. E...

Transforming growth factor beta (TGFb) regulates cell adhesion, proliferation, and differentiation in a variety of cells. Smad proteins are receptor-activated transcription factors that translocate to the nucleus in response to TGFb. We demonstrate here that TGFb increases cell adhesion in metastatic PC3-M prostate cancer cells. TGFb treatment of PC3-M cells leads to nuclear translocation of R-Smad proteins. We show that Smad proteins are necessary, but not sufficient, for TGFbmediated cell adhesion. After showing that TGFb upregulated p38 MAP kinase activity in PC3-M cells, we show that inhibition of p38 MAP kinase partially blocked TGFb-mediated increase in cell adhesion, as well as nuclear translocation of Smad3. Finally, we show that Smad3 is phosphorylated by p38 MAP kinase in vitro. These findings implicate crosstalk between the MAP kinase and Smad signaling pathways in TGFb's regulation of cell adhesion in human prostate cells. This represents a mechanism by which the pleiotropic effects of TGFb may be channeled to modulate cell adhesion.

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with fulllength reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors. (Journal of Biomolecular Screening 2008:755-765)