Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Recombinant Nup153 Incorporates in Vivo into Xenopus Oocyte Nuclear Pore Complexes

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.     

Detection of an intracellular transforming protein (v-Ki-ras p21) using the flow activated cell sorter (FACS)

Summary The transforming protein coded for by the Ki-ras oncogene, v-p21, localizes at the cytoplasmic face of the plasma membrane. A method is presented whereby the appearance of v-p21 in Kirsten murine sarcoma virustransformed cells can be detected by flow cytometry, using a monoclonal antibody to v-p21 and methods modified from immunofluorescence microscopy. The method is sufficiently sensitive to differentiate between cellular and viral p21 levels, to detect small subpopulations of virus-transformed cells, and to monitor changes in p21 expression in response to physiologic variables. The method provides a rapid, quantitative means to investigate the expression of an intracellular transforming protein in heterogeneous cell populations. Supported by studentships to D. F. from the B. C. Cancer Foundation, by a grant and a research associateship to N. A. from the National Cancer Institute of Canada, and by MRC grants ME 8456 and #68-7824 to the FACS committee at the University of British Columbia.     

Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.     

Unsuspected task for an old team: Succinate,fumarate and other Krebs cycle acids in metabolic remodeling

Seventy years from the formalization of the Krebs cycle as the central metabolic turntable sustaining the cell respiratory process, key functions of several of its intermediates, especially succinate and fumarate, have been recently uncovered. The presumably immutable organization of the cycle has been challenged by a number of observations, and the variable subcellular location of a number of its constitutive protein components is now well recognized, although yet unexplained. Nonetheless, the most striking observations have been made in the recent period while investigating human diseases, especially a set of specific cancers, revealing the crucial role of Krebs cycle intermediates as factors affecting genes methylation and thus cell remodeling. We review here the recent advances and persisting incognita about the role of Krebs cycle acids in diverse aspects of cellular life and human pathology.     

Influence of zinc on soluble carbohydrate and free amino acid levels in rapeseed plants regenerated in vitro in the presence of zinc

To gain more insight into the impact of zinc on the primary metabolites in rapeseed, plants were regenerated in vitro in the presence of zinc (0.1–1 mM), acclimatized, transferred to a greenhouse, and treated with 2 mM ZnSO₄. The main metabolites, including soluble carbohydrates and free amino acids, were analyzed by nuclear magnetic resonance spectroscopy, and further confirmed by spectrophotometry and enzymatic analyses. Exposure of these greenhouse-grown plants to ZnSO₄ led to both a significant rise of the total amino acid level and an increase of proline accumulation in the different tissues. Moreover, the control plants, regenerated without zinc stress, exhibited a significant reduction of soluble carbohydrate levels, whereas the plants derived from Zn-treated regenerants exhibited significantly higher levels of both glucose and sucrose. These levels increased proportionally with the Zn concentration in the regeneration step.     

Drosophilids (Diptera) from Mayotte island: an annotated list of species collected in 2013 and comments on the colonisation of Indian Ocean Islands

Summary. The Indian Ocean Islands are a most interesting region for evolutionary studies of terrestrial organisms and, among insects, the Drosophilidae family occupies a privileged position. The Comoros archipelago was, up to now, the least explored place among all the islands. We present here the results of a collection on one of the four main islands, Mayotte. From 4500 collected flies, 25 species were distinguished. The biology, ecology and biogeography of each species are discussed. Considering the extant known species from all islands, five evolutionary scenarios are proposed, ranging from the invasive, cosmopolitan, man-transported species to endemic species restricted to a single island. Some species raise a puzzling problem: despite having a very narrow and specialised ecological niche, they are broadly distributed on most islands and also on the African mainland.