RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

Spatial correlograms of soil cover as an indicator of landscape heterogeneity

Soil cover, which is one of the most informative and integrative landscape factors, can be used for the analysis of landscape patterns. We studied the spatial autocorrelation (Moran's I) of raster format soil maps (1:10,000; 10 m pixel size) in 35 study areas representing all landscape regions in Estonia. The carbonate concentration of soils, volumetric soil moisture (%) and the depth of the groundwater table were taken into consideration in compiling a scale of contrast of 17 soil groups. We introduce a simple characteristic based on spatial correlograms: a half-value distance lag, h_I = 0.5—a distance where Moran's I drops below 0.5. Spatial autocorrelation decreased very rapidly in the case of heights with a very heterogeneous landscape composition, showing low values of h_I = 0.5 (<100 m in all 6 study areas). In uplands and depressions, the spatial autocorrelation also decreased relatively rapidly (h_I = 0.5 < 200 m). In most of the plains, coastal lowlands, sea islands and inland paludified lowlands, the values of Moran's I did decrease slowly with increasing lag, being >200 m in all forest and bog areas with complex topographical conditions due to the variety of glacial landforms and peatlands. All of the eight FRAGSTATS landscape metrics studied demonstrated significant correlations with h_I = 0.5, whereas five of them – Contrast Weighted Edge Density (CWED); Percentage of Like Adjacencies (PLADJ), Edge Density (ED), Patch Density (PD) and Mean Patch Area Distribution (AREA_MN) – had Spearman Rank Order Correlation values higher than 0.8. Landscapes with high ED, PD, and CWED values have a low autocorrelation: PD, ED, and CWED correlated negatively with h_I = 0.5. PD, ED, and CWED decreased and PLADJ increased with the power-law relationship with increasing h_I = 0.5. Spatial autocorrelation is lower in landscapes with complex structure and high contrast. The positive relationship with PLADJ indicates the same. Thus, spatial correlograms of potential landscape structure based on soil cover analysis can be used for the characterization of human-influenced landscape (land use) structure.     

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

Background

Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.

Methods and Findings

Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB⁺ Tc cells) or granzyme A and granzyme B deficient (GzmA×B^−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.

Significance

Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.     

4-Aminobenzoic acid hydrazide inhibition of microperoxidase-11: catalytic inhibition by reactive metabolites

Inhibition of human peroxidase enzymes such as myeloperoxidase or eosinophil peroxidase represents a novel therapeutic area, for which there are no current clinical therapeutics. We utilized 4-aminobenzoic acid hydrazide which was reported to be a potent irreversible inhibitor of myeloperoxidase to gain insight into the role of reactive metabolites in catalytic inhibition. In order to carry out detailed studies, we used a model peroxidase, microperoxidase-11 (MP-11). We investigated the heme spectrum of MP-11 in the presence of 4-ABAH and found that heme bleaching occurred that was irreversible. This coincided with an absence of catalytic activity. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was able to significantly prevent inactivation of peroxidase activity, therefore, we performed ESR spin trapping studies and detected a carbonyl carbon-centered radical of 4-ABAH. In order to determine if the free radical metabolites became bound to MP-11, we performed high-resolution MALDI with elemental analysis to determine the change in elemental composition that occurred in these reactions. These masses were assigned to free radical metabolites of 4-ABAH and were not observed in reactions containing DMPO. We conclude that the 4-ABAH free radical metabolites which were bound to MP-11 were involved in the catalytic inhibition and were scavenged by DMPO.     

Identification,cloning, heterologous expression,and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from <Emphasis Type="Italic">Collinsella aerofaciens</Emphasis>

A gene encoding an NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) from Collinsella aerofaciens DSM 3979 (ATCC 25986, formerly Eubacterium aerofaciens) was identified and cloned in this study. Sequence comparison of the translated amino acid sequence suggests that the enzyme belongs to the short-chain dehydrogenase superfamily. This enzyme was expressed in Escherichia coli with a yield of 330 mg (5,828 U) per liter of culture. The enzyme catalyzes both the oxidation of ursodeoxycholic acid (UDA) forming 7-keto-lithocholic acid (KLA) and the reduction of KLA forming UDA acid in the presence of NADP⁺ or NADPH, respectively. In the presence of NADPH, 7β-HSDH can also reduce dehydrocholic acid. SDS-PAGE and gel filtration of the expressed and purified enzyme revealed a dimeric nature of 7β-HSDH with a size of 30 kDa for each subunit. If used for the oxidation of UDA, its pH optimum is between 9 and 10 whereas for the reduction of KLA and dehydrocholic acid it shows an optimum between pH 4 to 6. Usage of the enzyme for the biotransformation of KLA in a 0.5-g scale showed that this 7β-HSDH is a useful biocatalyst for producing UDA from suitable precursors in a preparative scale.