Deleted in Liver Cancer 2 (DLC2) Was Dispensable for Development and Its Deficiency Did Not Aggravate Hepatocarcinogenesis

DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.     

Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express

Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult-to-express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult-to-express antibody (mAb-A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb-A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb-A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point-mutant versions of difficult-to-express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.     

Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.     

Mutual regulation between mitochondrial ATPase and respiratory chain activities

Compounds made from the reaction of fluorescamine with simple primary amines and with mycosamine-containing macrolide antibiotics (e.g., amphotericin B) are used to investigate possible interactions between ATPase and respiration enzymes in rat liver mitochondria. The following observations have been made. (1) The acyclic form of the benzyl amine-fluorescamine compound stimulates the ATPase-linked inorganic phosphate formation, and this stimulation is not affected by rotenone, antimycin A, and potassium cyanide. In contrast, the respiratory inhibitors are able to prevent the stimulation of ATPase activity that is caused by conventional uncouplers e.g., 2,4-dinitrophenol. (2) The acyclic form of the amphotericin B-fluorescamine compound has no effect on ATPase-linked inorganic phosphate formation rate. However, in the presence of the antibiotic-fluorescamine compounds, the respiratory inhibitors are no longer able to prevent the uncoupler-stimulated ATPase activity. (3) The amine-fluorescamine modifiers have no effect on rotenone-sensitive NADH-cytochrome c reductase, on succinate-cytochrome c reductase, and on cytochrome oxidase in submitochondrial particles. (4) The amine-fluorescamine modifiers decrease the rate of the ATP-driven NAD⁺ reduction by succinate in submitochondrial particles. (5) The amine-fluorescamine modifiers inhibit the stimulation of respiration that is caused by conventional uncouplers, although the modifiers have no effect on the kinetics of the proton influx induced by uncouplers. The data are consistent with the hypothesis that the ATPase-linked and respiration-linked proton pumps may interact directly with each other, and this step establishes the mutual regulation between ATPase and respiratory activities.     

The catalytic architecture of leukotriene C4 synthase with two arginine residues

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).     

Tracking current and forecasting future land-use impacts of agricultural value chains. 67th Discussion Forum on Life Cycle Assessment, 3rd of November 2017, Zurich,Switzerland

The 67th Discussion Forum on Life Cycle Assessment (LCA), organised by partners of the European project RELIEF (RELIability of product Environmental Footprints), focused on methods for better understanding the impacts of land use linked to agricultural value chains. The first session of the forum was dedicated to methods that help in retrospective tracking of land use within complex supply chains. Novel approaches were presented for the integration of increasingly available spatially located land use data into LCA. The second session focused on forward-looking projections of land use change and included emerging, predictive methods for the modelling of land change. The third session considered impact assessment methods related to the use of land and their application together with land change modelling approaches. Discussions throughout the day centred on opportunities and challenges arising from integrating spatially located land use information into Life Cycle Assessment. Increasing amounts of spatially located land use data are becoming available and this could potentially increase the robustness and specificity of Life Cycle Assessment. However, the use of such data can be computationally expensive and requires the development of skills (i.e. use of geographical information systems (GIS) and model coding) within the LCA community. Land change modelling and ecosystem service modelling are associated with considerable uncertainty which must be communicated appropriately to stakeholders and decision-makers when interpreting results from an LCA. The new approaches were found to challenge aspects of the traditional LCA approach—particularly the division between the life cycle inventory and impact assessment and the assumption of linearity between scale and impacts when deriving characterisation factors. The presentations from the DF-67 are available for download (www.lcaforum.ch), and video recordings can be accessed online (http://www.video.ethz.ch/events/lca/2017/autumn/67th.html).     

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3⁺ bacteria were present within single-membrane phagosomes that are LAMP1⁺. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3⁺ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Resource conversion: a generalizable mechanism for resource-mediated positive species interactions

Positive species interactions are ubiquitous in natural communities, but the mechanisms through which they operate are poorly understood. One proposed mechanism is resource conversion – the conversion by a benefactor species of a resource from a resource state that is inaccessible to a potential beneficiary species into a resource state that is accessible. Such conversion often occurs as a byproduct of resource consumption, and sometimes in exchange for non-resource benefits to the benefactor species. At least five known classes of interactions, including both facilitative and mutualistic ones, may be classified as resource conversion interactions. We formulated a generalizable mathematical model for resource conversion interactions and examined two model variants that represent processing chain and nurse plant interactions. We examined the conditions under which these conformed to the stress-gradient hypothesis (SGH), which predicts increased interaction benefits in more stressful environments. These yielded four key insights: 1) resource conversion interactions can be positive (towards the resource recipient) only when facilitator-mediated resource conversion is more efficient than the baseline, spontaneous, facilitator-independent resource conversion; 2) the sign of resource conversion interaction outcomes never switches (e.g. from net positive to net negative) with changing levels of resource availability, when all other parameters are kept constant; 3) processing chain interactions at equilibrium can never be positive in a manner that conforms to the SGH; 4) nurse plant interactions can be positive and conform to the SGH, although the manner in which they do depends largely on how resource stress is defined, and the environmental supply rate of surface soil moisture. The first two insights are likely to be generalizable across all resource conversion interactions. The general agreement of the model with empirical studies suggest that resource conversion is the mechanism underlying the aforementioned interactions, and an ecologically meaningful way of classifying these previously unassociated positive species interactions.