Radiosensitization by Kinase Inhibition Revealed by Phosphoproteomic Analysis of Pancreatic Cancer Cells

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Highlights

• •Proteomes and phosphoproteomes of radiosensitive and radioresistant PDAC cell lines.
• •Common activation of DDR is proven by ATM activity on known and novel substrates.
• •Resistant cells bear raised NQO1 expression, actin dynamics including FAK activity.
• •Inhibitors of CHEK Rabusertib and FAK Defactinib radiosensitize PDAC cells.

     

Proteomic response of the marine ammonia-oxidising archaeon Nitrosopumilus maritimus to iron limitation reveals strategies to compensate for nutrient scarcity

Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.     

A comprehensive proteome map of the lipid-requiring nosocomial pathogen Corynebacterium jeikeium K411

Corynebacterium jeikeium is a lipid-requiring pathogen that is considered as part of the normal microflora of the human skin and associated with severe nosocomial infections. Systematic reference maps of the cytoplasmic, cell surface-associated, and extracellular proteome fractions of the clinical isolate C. jeikeium K411 were examined by 2-DE coupled with MALDI-TOF MS. A sum total of 555 protein spots were identified by PMF, corresponding to 358 different proteins that were classified into functional categories and integrated into metabolic pathways. The majority of the proteins were linked to housekeeping functions in energy production and translation and to physiological processes in amino acid, carbohydrate, nucleotide, and lipid metabolism. A complete enzymatic machinery necessary to utilize exogenous fatty acids by beta-oxidation was detected in the cytoplasmic proteome fraction. In addition, several predicted virulence factors of C. jeikeium K411 were identified in the cell surface-associated and extracellular subproteome, including the cell surface proteins SurA and SurB, the surface-anchored pilus subunits SapA and SapB, the surface-anchored collagen adhesin CbpA, the cholesterol esterase Che, and the acid phosphatase AcpA.     

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.     

CD8+ cells protect mice against reinfection with the intestinal parasite Eimeria falciformis

We investigated cellular immune responses of mice infected with the apicomplexan parasite Eimeria falciformis in order to characterise protective immune mechanisms and effector functions. Adoptive transfer experiments with mesenterial lymph node cells (MLNC) from immune donor mice were performed, and the oocyst output monitored after challenge infection. Phenotypical analysis by fluorescence cytometry and T cell proliferation assay showed that already from day four post infection E. falciformis-specific lymphocytes were present in the MLN. The frequency of parasite-specific, IFN-γ producing CD4+ and CD8+ cells increased in this period by 9.8% and 16.4%, respectively. Infection experiments with IFN-γ deficient mice revealed that IFN-γ is involved in resistance to primary and secondary infection. Transfer of total MLNC from immune donors reduced the oocyst output by 65–74%, as compared to the oocyst output of animals transferred with cells from naïve donors. Transfer of CD8+ cells inhibited parasite development resulting in a reduction of oocyst numbers by 42–64%, whereas CD4+ cells showed no influence on resistance to reinfection.     

Noninvasive Monitoring of Respiratory Viruses in Wild Chimpanzees

To diagnose respiratory disease among wild great apes, there is a need for noninvasive diagnostic methods. Therefore, we analyzed fecal samples from habituated chimpanzees from Taï National Park, Côte d’Ivoire. Samples had been collected during four distinct outbreaks: two with known aetiology (March 2004 and February 2006) and two with unknown aetiology (October 2004 and August 2005). Fecal samples were screened by polymerase chain reaction (PCR) for the presence of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV), two paramyxoviruses previously found in lung tissue of chimpanzees that died due to respiratory disease. In the March 2004 outbreak, 72% of the tested individuals were positive for HMPV, and during the 2006 epidemic, 25% tested HRSV-positive. In the outbreaks where no causative pathogen was previously known, fecal samples tested positive for either HRSV or HMPV, showing that reinfection occurred. Virus sequences were generated and compared with sequences previously found in tissue; nearly identical virus sequences in both tissue and fecal samples were found. These results demonstrate that fecal samples collected during outbreak times can be used for the diagnostic and phylogenetic analysis of HMPV and HRSV. Using such diagnostic tools, systematic noninvasive disease investigation of respiratory outbreaks in wild great apes becomes possible. The methods presented here may also be applied for the investigation of further acute diseases in great apes and other species.     

THE IMPORTANCE OF DIATOM CELL SIZE IN COMMUNITY ANALYSIS1

The large variation in size and shape in diatoms is shown by morphometric measurements of 515 benthic and pelagic diatom species from the Baltic Sea area. The largest mean cell dimension (mostly the apical axis) varied between 4.2 and 653 μm, cell surface area between 55 and 344,000 μm², and cell volume between 21 and 14.2 × 10⁶μm³. The shape‐related index, length to width ratio, was between 1.0 and 63.3 and the shape‐ and size‐related index, surface area to volume ratio, was between 0.02 and 3.13. Diatom community analysis by multivariate statistics is usually based on counts of a fixed number of diatom valves with species scores irrespective of cell size. This procedure underestimates the large species for two reasons. First, the importance of a species with higher cell volume is usually larger in a community. Second, larger species usually have lower abundances and their occurrence in the diatom counts is stochastic. This article shows that co‐occurring small and large diatom species can respond very differently to environmental constraints. Large epiphytic diatoms responded most to macroalgal host species and small epiphytic diatoms most to environmental conditions at the sampling site. Large epilithic diatoms responded strongly to salinity, whereas small epilithic diatoms did so less clearly. The conclusion is that different scale‐dependent responses are possible within one data set. The results from the test data also show that important ecological information from diatom data can be missed when the large species are neglected or underestimated.     

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus

BackgroundInfections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C⁺ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C⁺ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C⁺ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2C^wt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2C^del/del) and heterozygous (NKG2C^wt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C⁺ cells. We therefore analyzed the PUUV-mediated NKG2C⁺ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Methodology/Principal findingsNKG2C⁺ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C⁺ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2C^del and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2C^wt/del allele was significantly overrepresented, compared to the NKG2C^wt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2C^wt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2C^wt/wt NK cells.Conclusions/SignificanceOur results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C⁺ NK cell response, as reflected by NKG2C^wt/del variant, may be associated with a higher risk for a severe hantavirus infections.