Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Constitutive nuclear factor (NF)-kB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. B reast cancer is a disease of the mammary epithelium, which is composed of two major types of differentiated cells: luminal epithelial cells and basal or myoepithelial cells.(1) Recent studies have identified self-renewing pluripotent stem cells in mammary epithelium and suggest a model in which these stem cells could differentiate into the luminal-or basal-restricted lineages. Molecular taxonomic analyses of breast cancers by gene expression profiling have identified five breast cancer subtypes: luminal A, luminal B, basal-like, ERBB2-positive, and normal breast-like.(2) This classification is closely associated with the differentiation model of mammary epithelium. Luminal-and basallike breast cancer subtypes express genes characteristic of the two distinct types of epithelial cells. These subtypes show different clinical courses and responses to therapeutic agents. The basallike subtype has been associated with aggressive behavior and poor prognosis and typically does not express estrogen receptor (ER), progesterone receptor (PR), or ERBB2 ("triple-negative" phenotype).(3) Therefore, patients with basal-like subtype are unlikely to benefit from currently available targeted therapeutic strategies, such as hormone therapy and Herceptin (Roche, Basel, Switzerland). It is thus crucial to identify effective molecular targets for this subtype of breast cancer.Nuclear factor (NF)-κB transcription factors are important regulators of the genes necessary for innate and adaptive immune responses and for the survival and proliferation of certain cell types. The NF-κB family is composed of five different proteins, including RelA, RelB, c-Rel, and the precursor and processed products of the NFKB1 (p105/p50) and NFKB2 (p100/p52) genes.These proteins homodimerize and/or heterodimerize to form active transcription factors. Two distinct NF-κB pathways have been proposed: the classical pathway, which activates the RelA-p50 complex, and the alternative pathway, which activates the RelBp52 complex.(4) In normal cells, activation of the classical and alternative pathways is tightly regulated by inhibitor of NF-κB (IκB) family proteins and a p100 protein, respectively. Both NF-κB pathways are aberrantly activated and involved in tumor development in various cancers, including breast cancer.(5,6) Previous studies have revealed that hormone-independent breast cancer cells exhibit cons...

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by ;-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

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Larval settlement rates, genetic structure, and gene flow of broadcast-spawning (Acropora tenuis) and planula-brooding (Stylophora pistillata) corals (Scleractinia) were compared within a 500 km range in the Ryukyu Archipelago. We conducted a laboratory experiment to investigate planula settlement rates, and a broad sampling survey to determine genetic variation in both species in the Archipelago. In the laboratory experiment, the planulae of S. pistillata settled a few hours after release, while those of A. tenuis started to settle at least 4 d after the release of gametes. The survival rates and competency periods of larvae were higher and longer for A. tenuis than for S. pistillata. These results suggest that broader dispersal is more likely for A. tenuis than for S. pistillata. In the population genetic analysis, we measured local (2 stations in a region) and regional (Okinawa, Kerama and Yaeyama) patterns of genetic variation with allozyme electrophoresis. We also inferred the levels of gene flow in the 2 species. In the study area, gene flow (N e m) and genetic distance (D) were, respectively, higher and smaller for the spawner A. tenuis (N e m = 3.5 to 16.4, D = 0.028 to 0.187) than for the brooder S. pistillata (N e m = 0.9 to 1.5, D = 0.026 to 0.309). Therefore, the planulae settlement rates were well in agreement with gene flow. In addition, for both species, N e m between the Okinawa and Kerama regions (30 to 150 km apart; N e m = 9.4 to 22.5 in A. tenuis and 1.4 to 3.3 in S. pistillata) was higher than that between the Okinawa-Kerama and Yaeyama regions (up to 500 km apart; N e m = 3.1 to 9.4 in A. tenuis and 0.5 to 1.4 in S. pistillata). The results suggest that coral populations in the Kerama Island are a major source of the coral planulae needed for the recovery of both brooding and spawning coral communities around the Okinawa Islands, after the mass-bleaching event in 1998. KEY WORDS: Scleractinian coral · Reproductive mode · Competency · Gene flow · Larvae source · Ryukyu ArchipelagoResale or republication not permitted without written consent of the publisher Mar Ecol Prog Ser 256: 87-97, 2003 1985, , Harrison & Wallace 1990. This suggests that the dispersal potential of brooded planula is more restricted than that of planulae originating from spawning, because of the shorter pre-competency period in brooders versus spawners. Nevertheless, a previous comprehensive study of the population genetics of scleractinian corals does not fully support this hypothesis. Ayre & Hughes (2000) studied the population genetics of 5 brooding and 4 spawning coral species on the Great Barrier Reef (GBR) of Australia. They estimated that, in 3 of the 5 brooding species and all of the spawning species, larval dispersal was sufficient to maintain moderate to high levels of gene flow along the entire GBR. In contrast, they estimated that local populations of the remaining 2 brooding species (Stylophora pistillata and Seriatopora hystrix) were genetically more weakly connected than the other 7 species.I...