The polypore mushroom Irpex lacteus, a new causative agent of fungal infections

Irpex lacteus, a wood-decaying basidiomycete, was isolated from a pulmonary abscess of an immunosuppressed child. This medical strain was compared morphologically and by sequencing of the ribosomal intergenic spacers with specimens from both culture collections and herbarium desiccated material. The patient was treated successfully with amphotericin B.     

Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.     

The atheroprotective effect of 17beta-estradiol depends on complex interactions in adaptive immunity

Estradiol prevents fatty streak formation in chow-fed atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice. We previously reported that fatty streak development of immunodeficient ApoE(-/-)/recombination activating gene 2 (RAG-2(-/-)) double-deficient mice was insensitive to estradiol. In the present work, we demonstrate that the reconstitution of ApoE(-/-)/RAG-2(-/-) with bone marrow from immunocompetent ApoE(-/-)/RAG-2(+/+) mice restores the protective effect of estradiol on fatty streak constitution. We extended this demonstration to the model of low-density lipoprotein receptor-deficient mice, establishing the obligatory role of mature lymphocytes in this process. We then investigated whether the protective effect of estradiol was mediated by a specific lymphocyte subpopulation by studying the hormonal effect on fatty streak constitution in recently developed models of ApoE(-/-) mice deficient in selective T-lymphocyte subsets (either TCRalphabeta+, CD4+, CD8+, or TCRgammadelta+ lymphocytes) or B lymphocytes. In all these specifically immunodeficient mice, estradiol administration to ovariectomized mice conferred protection as in immunocompetent ApoE(-/-) mice, clearly demonstrating that no single lymphocyte subpopulation was specifically required for this effect. These results point to additional lymphocyte-dependent mechanisms such as modulating the interactions among lymphocytes and between lymphocytes and endothelial and/or antigen-presenting cells.     

Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor

Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAPK activation in PSTT, we established the first PSTT cell culture, IST-2, from a surgically resected PSTT. IST-2 cells expressed HLA-G and Mel-CAM but not E-cadherin, an immunophenotype characteristic of PSTT. IST-2 cells were highly motile and invasive in culture as compared to choriocarcinoma JEG-3 cells and normal extravillous trophoblastic cells. Based on wound assay, time-lapse videomicroscopy for cell tracking, and invasion chamber assays, we found that the motility and invasion of IST-2 cells were significantly reduced (P<0.01) after treatment with the MEK inhibitors CI-1040 and PD 59089, which prevent activation of MAPK. In contrast, neither compound had any effect on normal extravillous trophoblastic cells or JEG-3 cells. In conclusion, our findings demonstrate a functional role of MAPK activation in the motility and invasion of PSTT.     

Phenotypic characterization of human chondrocyte cell line C-20/A4: a comparison between monolayer and alginate suspension culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.     

Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network

The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the environment were included. A total of 708 structural open reading frames (ORFs) were accounted for in the reconstructed network, corresponding to 1035 metabolic reactions. Further, 140 reactions were included on the basis of biochemical evidence resulting in a genome-scale reconstructed metabolic network containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to approximately 16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared with Escherichia coli. The reconstructed metabolic network is the first comprehensive network for a eukaryotic organism, and it may be used as the basis for in silico analysis of phenotypic functions.     

Evaluation of a new cellulose sponge-tipped swab for microbiological sampling: a laboratory and clinical investigation

A new type of swab (Cellswab; Cellomeda, Turku, Finland), utilizing a highly absorbent cellulose viscose sponge material, was compared to some traditional swabs. The survival of 14 aerobic and 10 anaerobic and microaerophilic bacterial species in the Cellswab, two commercial swab transport systems (Copan, Brescia, Italy, and Orion Diagnostica, Espoo, Finland), and one Dacron swab (Technical Service Consultants Ltd. [TSC], Heywood, United Kingdom) was evaluated. Bacteria were suspended in broth, into which the swabs were dipped. The Cellswab absorbed 1.3 times more fluid and released 3.5 times more fluid upon plating than the other swabs. Aerobic bacteria were stored in dry tubes, the others in transport medium, at 4 degrees C and room temperature (RT), for up to 14 days. Swab samples were transferred to plates at 0, 1, 2, 4, 7, and 14 days. For 10 strains the Cellswab yielded > or =10% of the original CFU for longer than all the other swabs. In the clinical study, the ability of the Cellswab to detect beta-hemolytic streptococci from throat samples (n = 995) was compared to that of the TSC Dacron swab. The swabs performed equally, both when their samples were transferred to plates immediately and after storage for 1 day at 4 degrees C or RT. The changes in normal microbiota after storage were also similar. The Cellswab was found to perform at least as well as ordinary swabs. It was better at storing fastidious strains, and at keeping bacteria viable for long storage times; it might well be a useful replacement or complement to ordinary swabs.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.