DNA manipulators: caught in the act

DNA is a dynamic molecule that undergoes constant changes in the cell through interactions with numerous proteins. Several classes of enzyme are specialized in promoting DNA rearrangements, including site-specific recombinases, DNA helicases, transposases and DNA topoisomerases. Recent structures of protein-DNA reaction intermediates trapped in various states of DNA remodeling, complemented by biochemical and biophysical functional studies, have enhanced our understanding of their respective mechanistic pathways.     

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AIF4-and different types of heparin

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca²⁺ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60°C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45°C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.     

OCI-5/GPC3, a Glypican Encoded by a Gene That Is Mutated in the Simpson-Golabi-Behmel Overgrowth Syndrome, Induces Apoptosis in a Cell Line–specific Manner

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3–induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line– specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.     

Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

Four QTL in Rice Associated with Broad Spectrum Resistance to Blast Isolates from Rice and Barley

Pyricularia grisea is the most destructive and cosmopolitan fungal pathogen of rice and it can also cause disease on other agriculturally important cereals. We determined the number, location and interaction of quantitative trait loci (QTL) associated with resistance to P. grisea isolates obtained from rice (THL142 and THL222) and barley (TH16 and THL80) grown in Thailand. The isolates showed a spectrum of virulence when used to inoculate a series of differentials. We used a reference blast resistance mapping population of rice (IR64 × Azucena). IR64 was highly resistant, and Azucena was highly susceptible, to all four isolates. The numbers of resistant vs. susceptible progeny suggest that the resistance of IR64 is determined by two or three genes with additive effects. The correlation coefficients for all pairwise comparisons of disease severity were high and highest between barley isolates and between rice isolates. Four QTL were detected, one on each of the following chromosomes 2, 8, 9 and 10. IR64 contributed resistance alleles at three of the QTL (chromosomes 2, 8 and 9). Azucena contributed the resistance allele at the QTL on chromosome 10 in response to inoculation with isolate THL142. The results of the QTL analysis support interpretation of the phenotypic frequency distributions regarding the number of genes determining resistance to the four isolates in this population. Our results are novel in adding blast isolates from barley to the catalogue of pathogen specificities to which a gene, or genes, from IR64 confer resistance.     

Urban-Hazard Risk Analysis: Mapping of Heat-Related Risks in the Elderly in Major Italian Cities

BackgroundShort-term impacts of high temperatures on the elderly are well known. Even though Italy has the highest proportion of elderly citizens in Europe, there is a lack of information on spatial heat-related elderly risks.ObjectivesDevelopment of high-resolution, heat-related urban risk maps regarding the elderly population (≥65).MethodsA long time-series (2001–2013) of remote sensing MODIS data, averaged over the summer period for eleven major Italian cities, were downscaled to obtain high spatial resolution (100 m) daytime and night-time land surface temperatures (LST). LST was estimated pixel-wise by applying two statistical model approaches: 1) the Linear Regression Model (LRM); 2) the Generalized Additive Model (GAM). Total and elderly population density data were extracted from the Joint Research Centre population grid (100 m) from the 2001 census (Eurostat source), and processed together using “Crichton’s Risk Triangle” hazard-risk methodology for obtaining a Heat-related Elderly Risk Index (HERI).ResultsThe GAM procedure allowed for improved daytime and night-time LST estimations compared to the LRM approach. High-resolution maps of daytime and night-time HERI levels were developed for inland and coastal cities. Urban areas with the hazardous HERI level (very high risk) were not necessarily characterized by the highest temperatures. The hazardous HERI level was generally localized to encompass the city-centre in inland cities and the inner area in coastal cities. The two most dangerous HERI levels were greater in the coastal rather than inland cities.ConclusionsThis study shows the great potential of combining geospatial technologies and spatial demographic characteristics within a simple and flexible framework in order to provide high-resolution urban mapping of daytime and night-time HERI. In this way, potential areas for intervention are immediately identified with up-to-street level details. This information could support public health operators and facilitate coordination for heat-related emergencies.