A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.     

Patterns of livestock depredation and cost‐effectiveness of fortified livestock enclosures in northern Tanzania

Human–carnivore conflicts and retaliatory killings contribute to carnivore populations' declines around the world. Strategies to mitigate conflicts have been developed, but their efficacy is rarely assessed in a randomized case–control design. Further, the economic costs prevent the adoption and wide use of conflict mitigation strategies by pastoralists in rural Africa. We examined carnivore (African lion [Panthera leo], leopard [Panthera pardus], spotted hyena [Crocuta crocuta], jackal [Canis mesomelas], and cheetah [Acinonyx jubatus]) raids on fortified (n = 45, total 631 monthly visits) and unfortified (traditional, n = 45, total 521 monthly visits) livestock enclosures (“bomas”) in northern Tanzania. The study aimed to (a) assess the extent of retaliatory killings of major carnivore species due to livestock depredation, (b) describe the spatiotemporal characteristics of carnivore raids on livestock enclosures, (c) analyze whether spatial covariates influenced livestock depredation risk in livestock enclosures, and (d) examine the cost‐effectiveness of livestock enclosure fortification. Results suggest that (a) majority of boma raids by carnivores were caused by spotted hyenas (nearly 90% of all raids), but retaliatory killings mainly targeted lions, (b) carnivore raid attempts were rare at individual households (0.081 raid attempts/month in fortified enclosures and 0.102 raid attempts/month in unfortified enclosures), and (c) spotted hyena raid attempts increased in the wet season compared with the dry season, and owners of fortified bomas reported less hyena raid attempts than owners of unfortified bomas. Landscape and habitat variables tested, did not strongly drive the spatial patterns of spotted hyena raids in livestock bomas. Carnivore raids varied randomly both spatially (village to village) and temporally (year to year). The cost‐benefit analysis suggest that investing in boma fortification yielded positive net present values after two to three years. Thus, enclosure fortification is a cost‐effective strategy to promote coexistence of carnivores and humans.     

Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

Interactor-mediated nuclear translocation and retention of protein phosphatase-1

Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.     

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

Background  

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC.     

Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53

Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53^+/+ HCT116 and p53^?/? H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC₅₀ values in the low micromolar range with a clearly more pronounced effect on the p53^+/+ HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules.     

Fab is the most efficient format to express functional antibodies by yeast surface display

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.     

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.     

Alteration of arachidonic acid metabolism with spleen microsomes of irradiated rats

Arachidonic acid is metabolized by a rat spleen microsomes cyclooxygenase into prostaglandin D₂, thromboxane B₂, 12-hydroxy-5, 8, 10-heptadecadienoic acid and by a lipoxygenase into 12-hydroxy-5, 8, 10, 14-eicosatetraenoic acid and other unidentified compounds as analyzed by a radiometric thin-layer chromatography method and by gas-chromatography-mass spectrometry. This conversion is modified when spleen microsomes are obtained from whole body irradiated rats. Furthermore, if exogenous cofactors are added to the incubation medium, other changes appear that are different for the lipoxygenase and the cyclooxygenase activities. The results suggest a regulatory role of cofactors on both enzymes and/or a modification of sensitivity of the microsomal fraction from irradiated rats to effectors.