SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Non-thermal atmospheric pressure plasma (NTAPP) is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS). However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO) did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells.

Abstract. A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the rasl signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2-phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with rasl. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of rasl and scdl, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for rasl for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdcl5, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the 13-tubulin gene, which arrests cells at the metaphase-anaphase transition. T Hc~ ras proteins are GTPases that cycle between anive, GTP-bound form and an inactive, GDP-.It. bound form (Boguski and McCormick, 1993). Depending on the cellular context, activated ras can stimulate the cell division cycle, alter cell shape, or cause cellular differentiation (Bourne et al., 1990). Several pathways are implicated in signaling downstream of mammalian ras proteins. The best characterized pathway activates the raf kinase. Activated ras recruits the raf kinase and activated raf, in turn, activates a mitogen-activated protein (MAP) 1 kinase cascade (Herskowitz, 1995;Marshall, 1995). A second ras effector may be phosphatidylinositol(PI)-3 kinase. Activated ras binds to PI-3 kinase and ras binding stimulates PI-3 kinase activity four-fold in vitro (RodriguezVinciana et al., 1994). An activated allele of PI-3 kinase, however, activates ras and requires ras for signaling, suggesting ras is an effector of PI-3 kinase (Hu et al., 1995). A third ras effector may be ralGDS, a positive regulator of the ral GTPase (Hofer et al., 1994;Kikuchi et al., 1994;Spaargaren and Bischoff, 1994). Activated ral GTPase Address all correspondence to Charles F. Albright, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, 343-2174; Fax: (615) 343-0704. 1. Abbreviations used in this paper: DAPI,...

Right lobe living-donor liver transplantation (LDLT) is often not attempted in donors with anomalous portal venous branching (APVB). The authors describe their experience with portal vein (PV) reconstruction in 17 cases of APVB in right lobe LDLT. From July 1997 to December 2001, 214 right liver LDLT were performed at the Asan Medical Center. Seventeen of the donors had APVB and successfully underwent right lobectomy. The APVB were type II (trifurcation) in nine cases, type III (independent posterior segmental branching from main PV trunk) in seven, and unclassified in one. All 17 donors and recipients are alive, with good liver function. In type II APVB, the donor PV branches were obtained with separate openings that were joined as a common orifice at the back table in two, with a discoid-patch single opening in four, and with one common opening in three. In type III APVB, the donor PV were divided with two openings in four and with a discoid-patch single opening in three. The discoid-patch defect in the remnant PV was repaired with a vein patchplasty in two donors and resected with end-to-end anastomosis in five. However, one donor developed portal vein thrombosis (PVT) that was managed successfully by re-exploration and insertion of a metallic vascular stent. Of the four type III APVB obtained with two separate PV openings, the first two liver grafts were each reconstructed as double PV anastomoses. One of them required re-exploration because of PVT. In the two succeeding cases, a Y-graft interposition technique using a cryopreserved cadaveric iliac vein or the recipient's own portal confluence was successfully applied. To minimize the risk of PVT in donors with APVB, discoid-patch excision followed by repair with vein patchplasty or segmental resection should be avoided. Individual division of the PV branches creating two separate openings instead is recommended. To decrease the recipient's risk of PVT, interposition Y-graft venous reconstruction at the back table is superior to double PV anastomoses.

The heat shock protein HSP70 plays antiapoptotic and oncogenic roles, and thus its inhibition has been recognized as a potential avenue for anticancer therapy. Here we describe the small molecule, apoptozole (Az), which inhibits the ATPase activity of HSP70 by binding to its ATPase domain and, as a result, induces an array of apoptotic phenotypes in cancer cells. Affinity chromatography provides evidence that Az binds HSP70 but not other types of heat shock proteins including HSP40, HSP60, and HSP90. We also demonstrate that Az induces cancer cell death via caspase-dependent apoptosis by disrupting the interaction of HSP70 with APAF-1. Animal studies indicate that Az treatment retards tumor growth in a xenograft mouse model without affecting mouse viability. These studies suggest that Az will aid the development of new cancer therapies and serve as a chemical probe to gain a better understanding of the diverse functions of HSP70.