Clinicopathological significance of MMP-2 and its specific inhibitor TIMP-2 in gastric cancer

Matrix metalloproteinases (MMPs) can degrade type IV collagen of extracellular matrices and basal membranes and thus play a key role in the migration of malignant cells. In vivo, MMPs are inhibited by tissue inhibitors of metalloproteinases (TIMPs). Since in a previous study we showed that the expression of MMP-2 correlates with clinicopathological parameters in gastric cancer, we have now investigated a possible correlation of MMP-2 and TIMP-2 expression with survival in gastric cancer, as well as the possible association of TIMP-2 with clinicopathological parameters. Tissue samples were obtained from 116 gastric cancer patients who underwent gastrectomy with extended lymphadenectomy. MMP-2 and TIMP-2 expression was analysed using immunohistochemical staining and was graded semiquantitatively (score 0 - 3). High epithelial MMP-2 immunoreactivity was significantly associated with tumor stage and poor survival using the Kaplan-Meier log-rank statistical method (log-rank statistics). However, using Cox regression analysis, high epithelial MMP-2 immunoreactivity was not an independent prognostic factor. TIMP-2 showed no association with survival in gastric cancer, but the intensity of TIMP-2 staining in tumor cells correlated significantly with tumor differentiation based on the WHO and Lauren and Ming classifications, as well as with presence of distant metastasis. Our results show that high epithelial MMP-2 expression in gastric cancer is associated with poor survival, although it is not an independent prognostic factor, and that aggressive forms of gastric cancer are associated with low TIMP-2 expression.     

Selective translation initiation on bicistronic simian virus 40 late mRNA.

We described previously a simian virus 40 (SV40) mutant, pSVAdL, that was defective in synthesis of the late viral protein VP1. This mutant, which contains a 100-base-pair fragment of adenovirus DNA encompassing the major late promoter inserted in the SV40 late promoter region (SV40 nucleotide 294), efficiently synthesizes agnoprotein, a protein encoded by the leader region of the same mRNA that encodes VP1. When the agnoprotein AUG initiation codon in pSVAdL was mutated to UUG, agnoprotein synthesis was abolished, and VP1 synthesis was elevated to wild-type levels. Because levels of late mRNA synthesis were not affected by this mutation, these results support a scanning model of translation initiation and suggest that internal translational reinitiation does not occur efficiently in this situation.     

Contributions of five secondary metal uptake systems to metal homeostasis of Cupriavidus metallidurans CH34

Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA(1) expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA(1) as the main secondary magnesium uptake system, and CorA(2) and CorA(3) as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.     

Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis

Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.     

Mechanisms of contact-mediated killing of yeast cells on dry metallic copper surfaces

Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC(2)(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.     

Additive effects of exotic plant abundance and land-use intensity on plant–pollinator interactions

The continuing spread of exotic plants and increasing human land-use are two major drivers of global change threatening ecosystems, species and their interactions. Separate effects of these two drivers on plant–pollinator interactions have been thoroughly studied, but we still lack an understanding of combined and potential interactive effects. In a subtropical South African landscape, we studied 17 plant–pollinator networks along two gradients of relative abundance of exotics and land-use intensity. In general, pollinator visitation rates were lower on exotic plants than on native ones. Surprisingly, while visitation rates on native plants increased with relative abundance of exotics and land-use intensity, pollinator visitation on exotic plants decreased along the same gradients. There was a decrease in the specialization of plants on pollinators and vice versa with both drivers, regardless of plant origin. Decreases in pollinator specialization thereby seemed to be mediated by a species turnover towards habitat generalists. However, contrary to expectations, we detected no interactive effects between the two drivers. Our results suggest that exotic plants and land-use promote generalist plants and pollinators, while negatively affecting specialized plant–pollinator interactions. Weak integration and high specialization of exotic plants may have prevented interactive effects between exotic plants and land-use. Still, the additive effects of exotic plants and land-use on specialized plant–pollinator interactions would have been overlooked in a single-factor study. We therefore highlight the need to consider multiple drivers of global change in ecological research and conservation management.     

Conundrums in mixed woody–herbaceous plant systems

Aims To identify approaches to improve our understanding of, and predictive capability for, mixed tree–grass systems. Elucidation of the interactions, dynamics and determinants, and identification of robust generalizations that can be broadly applied to tree–grass systems would benefit ecological theory, modelling and land management. Methods A series of workshops brought together scientific expertise to review theory, data availability, modelling approaches and key questions. Location Ecosystems characterized by mixtures of herbaceous and woody plant life‐forms, often termed ‘savannas’, range from open grasslands with few woody plants, to woodlands or forests with a grass layer. These ecosystems represent a substantial portion of the terrestrial biosphere, an important wildlife habitat, and a major resource for provision of livestock, fuel wood and other products. Results Although many concepts and principles developed for grassland and forest systems are relevant to these dual life‐form communities, the novel, complex, nonlinear behaviour of mixed tree–grass systems cannot be accounted for by simply studying or modelling woody and herbaceous components independently. A more robust understanding requires addressing three fundamental conundrums: (1) The ‘treeness’ conundrum. What controls the relative abundance of woody and herbaceous plants for a given set of conditions at given site? (2) The coexistence conundrum. How do the life‐forms interact with each other? Is a given woody–herbaceous ratio dynamically stable and persistent under a particular set of conditions? (3) The net primary productivity (NPP) conundrum. How does NPP of the woody vegetation, the herbaceous vegetation, and the total ecosystem (woody + herbaceous) change with changes in the tree–grass ratio? Tests of the theory and conceptual models of determinants of mixed woody–herbaceous systems have been largely site‐ or region‐specific and have seldom been broadly or quantitatively evaluated. Cross‐site syntheses based on data and modelling are required to address the conundrums and identify emerging patterns, yet, there are very few data sets for which either biomass or NPP have been quantified for both the woody and the herbaceous components of tree–grass systems. Furthermore, there are few cross‐site comparisons spanning the diverse array of woody–herbaceous mixtures. Hence, initial synthesis studies should focus on compiling and standardizing a global data base which could be (1) explored to ascertain if robust generalizations and consistent patterns exist; and (2) used to evaluate the performance of savanna simulation models over a range of woody–herbaceous mixtures. Savanna structure and productivity are the result of complex and dynamic interactions between climate, soils and disturbances, notably fire and herbivory. Such factors are difficult to isolate or experimentally manipulate in order to evaluate their impacts at spatial and temporal scales appropriate for assessing ecosystem dynamics. These factors can, however, be evaluated with simulation models. Existing savanna models vary markedly with respect to their conceptual approach, their data requirements and the extent to which they incorporate mechanistic processes. Model intercomparisons can elucidate those approaches most suitable for various research questions and management applications. Conclusion Theoretical and conceptual advances could be achieved by considering a broad continuum of grass–shrub–tree combinations using data meta‐analysis techniques and modelling.