Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.     

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b₅ anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb₅ anchor (1–266-b₅) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb₅ anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

The perceived temperature – a versatile index for the assessment of the human thermal environment. Part A: scientific basics

The Perceived Temperature (PT) is an equivalent temperature based on a complete heat budget model of the human body. It has proved its suitability for numerous applications across a wide variety of scales from micro to global and is successfully used both in daily forecasts and climatological studies. PT is designed for staying outdoors and is defined as the air temperature of a reference environment in which the thermal perception would be the same as in the actual environment. The calculation is performed for a reference subject with an internal heat production of 135 W m⁻² (who is walking at 4 km h⁻¹ on flat ground). In the reference environment, the mean radiant temperature equals the air temperature and wind velocity is reduced to a slight draught. The water vapour pressure remains unchanged. Under warm/humid conditions, however, it is implicitly related to a relative humidity of 50%. Clothing is adapted in order to achieve thermal comfort. If this is impossible, cold or heat stress will occur, respectively. The assessment of thermal perception by means of PT is based on Fanger’s Predicted Mean Vote (PMV) together with additional model extensions taking account of stronger deviations from thermal neutrality. This is performed using a parameterisation based on a two-node model. In the cold, it allows the mean skin temperature to drop below the comfort value. In the heat, it assesses additionally the enthalpy of sweat-moistened skin and of wet clothes. PT has the advantages of being self-explanatory due to its deviation from air temperature and being—via PMV—directly linked to a thermo-physiologically-based scale of thermal perception that is widely used and has stood the test of time. This paper explains in detail the basic equations of the human heat budget and the coefficients of the parameterisations.     

Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.     

Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.