Asymmetric Jetting during the Impact of Liquid Drops on Superhydrophobic Concave Surfaces.

The impact of liquid drops on superhydrophobic solid surfaces is ubiquitous and of practical importance in many industrial processes. Here, we study the impingement of droplets on superhydrophobic surfaces with a macroscopic dimple structure, during which the droplet exhibits asymmetric jetting. Systematic experimental investigations and numerical simulations provide insight into the dynamics and underlying mechanisms of the observed phenomenon. The observation is a result of the interaction between the spreading droplet and the dimple. An upward internal flow is induced by the dimple, which is then superimposed on the horizontal flow inside the spreading droplet. As such, an inclined jet is issued asymmetrically into the air. This work would be conducive to the development of an open-space microfluidic platform for droplet manipulation and generation.

COBRA1 inhibits AP-1 transcriptional activity in transfected cells.

Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis.

BRCA1 interacts with FHL2 and enhances FHL2 transactivation function.

Germ-line mutations in BRCA1 are associated with an increased lifetime risk of developing breast and/or ovarian tumors. The BRCA1 gene product is a 220-kDa protein that contains a tandem of two BRCA1 C-terminal (BRCT) domains required for transcription. In an attempt to understand how BRCA1 exerts its function through BRCT domains, we search for partners of the BRCT domains of BRCA1. Using the yeast two-hybrid system, we identified the four and a half LIM-only protein 2 (FHL2) as a novel BRCA1 interacting protein. We demonstrate that BRCA1 and FHL2 can physically associate in vitro, in yeast, and in human cells. BRCA1 interacted with FHL2 through its second BRCT domain and the interaction of FHL2 with BRCA1 requires the last three LIM domains of FHL2. BRCA1 enhanced FHL2-mediated transcriptional activity in transient transfections. Tumor-derived transactivation-deficient BRCA1 mutants showed a reduced ability to enhance transactivation by FHL2. Lack of BRCA1 binding sites in the FHL2 completely abolished the FHL2 transactivation function. Reverse transcription polymerase chain reaction analysis showed that FHL2 mRNA levels may be downregulated in many breast cancer cell lines. These results suggest that the BRCA1-FHL2 interaction may be involved in transcriptional regulation and play a significant role in cancer cell growth.

Ligand-independent activation of estrogen receptor alpha by XBP-1.

The estrogen receptor (ER) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that XBP-1 mRNA expression is associated with ERalpha status in breast tumors. However, the role of XBP-1 in ERalpha signaling remains to be elucidated. More recently, two forms of XBP-1 were identified due to its unconventional splicing. We refer to the spliced and unspliced forms of XBP-1 as XBP-1S and XBP-1U, respectively. Here, we report that XBP-1S and XBP-1U enhanced ERalpha-dependent transcriptional activity in a ligand-independent manner. XBP-1S had stronger activity than XBP-1U. The maximal effects of XBP-1S and XBP-1U on ERalpha transactivation were observed when they were co-expressed with full-length ERalpha. SRC-1, the p160 steroid receptor coactivator family member, synergized with XBP-1S or XBP-1U to potentiate ERalpha activity. XBP-1S and XBP-1U bound to the ERalpha both in vitro and in vivo in a ligand-independent fashion. XBP-1S and XBP-1U interacted with the ERalpha region containing the DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, including the N-terminal basic region leucine zipper domain (bZIP) and the C-terminal activation domain. The bZIP-deleted mutants of XBP-1S and XBP-1U completely abolished ERalpha transactivation by XBP-1S and XBP-1U. These findings suggest that XBP-1S and XBP-1U may directly modulate ERalpha signaling in both the absence and presence of estrogen and, therefore, may play important roles in the proliferation of normal and malignant estrogen-regulated tissues.

[XBP-1 enhances the transcriptional activity of estrogen receptor alpha].

The estrogen receptor (ERalpha) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that human X-box binding protein 1 (XBP-1) mRNA expression is associated with ERalpha status in breast tumors. More recently, two forms of XBP-1 were identified due to their unique splicing. The two splicing variants of XBP-1 were designated XBP-1S and XBP-1U, respectively. In this study, the coding sequences of XBP-1S and XBP-1U were cloned respectively into the expression vector pcDNA3 harboring FLAG epitope, generating the recombinant plasmids pcDNA3-FLAG-XBP-1S and pcDNA3-FLAG-XBP-1U. Western blot analysis showed that both XBP-1S and XBP-1U were expressed in mammalian cells. To determine the effects of XBP-1S and XBP-1U on the transcriptional activity of ERalpha, MDA-MB-231 breast cancer cells were cotransfected with the expression vectors for ERalpha and either pcDNA3-FLAG-XBP-1S or pcDNA3-FLAG-XBP-1U. The results indicated that XBP-1S and XBP-1U enhanced ERalpha-mediated transcriptional activities in a hormone-independent manner. GST pull-down assay showed that both XBP-1S and XBP-1U interacted with ERalpha. These data suggest that XBP-1S and XBP-1U may play an important role in breast cancer growth and progression through ERalpha signaling.

[Construction of ERbeta expression vector and its function in different cancer cells].

OBJECTIVE: To construct an ERbeta expression vector and study its expression and function in different cancer cells. METHODS: Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements. RESULTS: The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3. CONCLUSION: ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.

[Isolation and characterization of a BRCA1-interacting protein].

BRCA1 (breast cancer susceptibility gene-1) plays important roles in DNA damage repair, cell checkpoint regulation, gene transcription, chromosome stability, and apoptosis. At the C-terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2). In this study, to identify proteins that interact with the BRCT2 domain of BRCA1, the standard yeast two-hybird screen was performed. FHL2 was isolated from a human ovary library, with the BRCT2 domain of BRCA1 as bait. Furthermore, the specific interaction of FHL2 with the BRCT2 domain of BRCA1, but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1, was verified by yeast mating. To confirm the interaction between BRCA1 and FHL2 in vitro, the GST pull-down assay was performed, the coding sequences of BRCT1 and BRCT2 domains were fused in-frame with the coding region of GST in the pGEX-2T vector, generating the pGST-BRCT1 and pGST-BRCT2 recombinant plasmids the fusion proteins GST-BRCT1 and GST-BRCT2 were expressed in E. coli DH5 alpha. The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography. The purified fusion proteins were incubated with in vitro translated 35S-methinine-labeled FHL2. Consistent with the two-hybird results, FHL2 could specifically bind to the BRCT2 domain, but not BRCT1 in vitro. To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo, pFLAG-FHL2 and pHABRCT1/pHA-BRCT2 recombinant plasmids were cotransfected into 293T cells. Then the coimmunoprecipitation assay were performed. The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo. Furthermore, the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo. These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression.

[Mapping of BRCT1 domain of BRCA1 with chromatin unfolding activity].

Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.

[Studies of mutations in BRCA1 transactivation domain by visualization of chromatin structure].

Mutations in breast cancer susceptibility gene 1(BRCA1) account for approximately 40%-50% of familial breast cancer cases and for more than 80% of inherited breast and ovarian cancer cases. Many cancer-predisposing mutations are located in the C-terminal region that functions as a transcriptional activation domain, but most of the mutations in the transactivation domain identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. Because chromatin structure regulation is an early event in gene transcription control, the chromatin unfolding activities of different transactivation domain variants were compared with that of the wild-type transactivation domain by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To do this, different constructs of the transactivation domain were selected as follows: (a) the wild-type transactivation domain; (b) two polymorphisms (S1613G and M1652I); and (c) four cancer-predisposing mutations (A1708E, M1775R, W1837R and Y1853 term). All of the constructs were made by fusing in frame with lac repressor. Western blot analysis indicated that all of the fusion proteins were expressed in A03 1 cells, in which multiple copies of the lac operator were integrated to produce a heterochromatic region of the genome. The chromatin unfolding assay showed that, like the wild-type transactivation domain, two variants that represent benign polymorphisms did not induce chromatin unfolding or only induced subtle change. Contrary to the behaviors of the wild type and two benign variants, four cancer-predisposing mutations in the transactivation domain superactivate the chromatin unfolding. The results suggest that the chromatin unfolding assay can aid in the characterization of deterious mutations in the C-terminal transactivation domain of BRCA1 and may provide more reliable presymptomatic risk assessment.