Human ORFeome Version 1.1: A Platform for Reverse Proteomics

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.     

c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression

c-Myc promotes cell growth and transformation by ill-defined mechanisms. c-myc(-/-) mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. Here we report that the lethality of c-myc(-/-) embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, c-myc(-/-) embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), as VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, c-myc(-/-) ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors.     

Organization and expression of human telomere repeat binding factor genes

The ends of mammalian chromosomes terminate in structures called telomeres. Recently a human telomere repeat binding factor (TRF1) that binds the vertebrate TTAGGG telomeric repeat in situ was isolated by Chong et al. (1). TRF1 regulates telomere length (2), which is often altered in cancer cells. To understand their genetic organization, TRF1 genes were localized to human chromosomes 13 cen, 21cen, and Xq13 by analysis of human monochromosomal hybrids, and by fluorescent in situ hybridization. We also confirmed the recent localization of a human TRF1 gene to chromosome 8, and provide evidence that this locus is alternatively spliced. In contrast to the TRF1 genes on chromosomes 8 and X, the chromosomes 13 and 21 TRF1 genes contained a 60 bp deletion in the coding region. The results suggest that two distinct forms of TRF1 are expressed and that the TRF1 gene family includes at least three pseudogenes whose dispersal in the human genome may have occurred via cDNA intermediates.     

The academic elite in health services administration: linkages among top-ranked graduate programs

The eleven top-ranked graduate programs in health services administration, based on a national survey of deans, top administrators, and senior faculty, were linked to one another by hiring one another's graduates. It is suggested that this linkage helps these programs maintain and enhance their prestige.     

Analysis of dispensing activities before and after decentralization of pharmaceutical services

Changes in drug handling activities, revenue, and telephone communications were documented during a conversion from a centralized unit dose system to decentralized pharmacists and unit dose services in a 310-bed university teaching hospital. All decentralized services were mobile; no physical satellites were utilized. Computer programs were used to collect and analyze drug handling and revenue data during a prestudy control period and three equal-length study periods after decentralization of pharmaceutical services for five patient care areas of the hospital. All telephone calls to the central pharmacy were recorded and classified by type during 21 days of the prestudy period and were compared with 21 days of the second postimplementation period. The mean number of doses handled decreased for all patient care areas. After decentralization the number of telephone calls to the central pharmacy requesting clinical drug information, as well as distributive information, decreased sharply. Moving the pharmacist to the patient care unit decreased the time that pharmacists spent handling drugs and improved communication with the medical and nursing staffs.     

An efficient multiple-exposure analysis of the toxicity of crisnatol,a DNA intercalator in phase II clinical trials

To investigate the toxicity and mechanism of action of crisnatol (CRS), a new DNA intercalator currently in phase II clinical trials, we analyzed cellular and nuclear flow cytometric (FCM) parameters of murine erythroleukemic cells (MELC) exposed to a range of CRS concentrations over three exposure conditions: short-term (4 h), long-term (24 h), and short-term with recovery (4 h⁺/19 h^–). At 0.5–1.0M CRS, 4 h exposure results in a reversible G₂-phase block, while 24 h exposure results in > G₂ polyploidy. At 5–10M CRS concentrations, cells exhibit persistent retardation of S-phase progression or irreversible G₂ and/or > G₂ blocks, depending on duration of exposure. Cells terminally blocked in G₂ exhibit increased nuclear/cellular volumes and increased nuclear fluorescein isothiocyanate (protein) staining, suggestive of unbalanced growth. At 25–50M CRS concentrations, MELC exhibit severe membrane perturbation (loss of viability) regardless of exposure. In contrast, following similar exposures to an inactive isomer of CRS, MELC exhibit minimal cell cycle effects, suggesting that cell cycle kinetics may be a useful criterion for assessing potential efficacy. Similar analyses with different classes of chemotherapeutic agents reveal that the range of induced cellular/nuclear perturbations varies with the class of compound used. Taken together, these results suggest that drug toxicity can vary with both concentration and duration of exposure and, as such, a selective multiple-exposure FCM analysis may better represent the spectrum of drug action for drug development and pharmacodynamic studies.Abbreviations m-AMSA amsacrine - ARA-C cytosine arabinoside - BrdU 5-bromodeoxyuridine - CF 5,6-carboxyfluorescein - CFDA 5,6-carboxyfluorescein diacetate - CRS crisnatol - FCM flow cytometry - FITC fluorescein isothiocyanate - 5-FU 5-fluorouracil - DMSO dimethylsulfoxide - MELC Friend murine erythroleukemic cell - MLP melphalan - PBS phosphate-buffered saline - PI propidium iodide - TAX taxol - topo topoisomerase - VBL vinblastine sulfate - cis-Pt cis-platinum