Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a specialized type III secretion (TTS) system into the host cell cytosol. The efficient secretion of many effector proteins depends on the global TTS chaperone HpaB. Here, we identified a novel export control protein, HpaC, which significantly contributes to bacterial pathogenicity. Deletion of hpaC leads to a severe reduction in secretion of effector proteins and the putative type III translocon proteins HrpF and XopA. By contrast, secretion of the TTS pilus protein HrpE is not affected. We provide experimental evidence that HpaC differentiates between two classes of effector proteins. Using an in vivo reporter assay, we found that HpaC specifically promotes the translocation of the effector proteins XopJ and XopF1 into the plant cell, whereas AvrBs3 and XopC are efficiently translocated even in the absence of HpaC. Similar findings were obtained for HpaB. Inhibition of protein synthesis suggests that HpaB is involved in the secretion of stored effector proteins. Furthermore, protein-protein interaction studies revealed that HpaB and HpaC form an oligomeric protein complex and that they interact with members of both effector protein classes and the conserved TTS system component HrcV. Taken together, our data indicate that HpaB and HpaC play a central role in recruiting TTS substrates to the secretion apparatus.     

Identification of intracellular proteins associated with the EBV-encoded nuclear antigen 5 using an efficient TAP procedure and FT-ICR mass spectrometry

Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and alpha- and beta-tubulin.     

α1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro

Background  

Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor α-1 antitrypsin (AAT) or its C-terminal fragment (C-36 peptide), on cultured lung cancer cells.     

Cellular roles of ADAM12 in health and disease

ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.     

Hybridization among Tilia cordata and T. platyphyllos (Tiliaceae) in Denmark

Morphological evidence of hybridization among Tilia cordata and T. platyphyllos is presented in plots of principal components analysis (PCA) and Wells' distance diagrams, the latter based on differently weighted characters. Putative hybrids from both natural and planted stands in Denmark are studied. Plausible hybrids found by running Estabrook's HYWIN program on the data are almost identical with those found by PCA and the weighted Wells' distance coefficient.     

The Ecology of Scale: Assessment of Regional Energy Turnover and Comparison with Global Food (5 pp)

Goal, Scope and Background Obviously, people assume that the regional production and distribution of food requires less energy turnover, compared with global transports of food. Politicians claim the term “regionality”, maintaining that regionality is a medicine against wasting so much energy for the global food distribution. Additionally the energy turnover is causing pollution. But remarkably, there is a lack of empirical data to support this idea. At the same time, nobody really requires regional origin for non-food items, e.g. for bicycles, dishwashers, furniture or cars etc. The public mainstream asks for regional origin merely for food. Hence, the first scientific issue worked out in this paper is how the energy turnover of comparable food items can be measured, regarding the lifecycle of food in total. That means to investigate the partial systems of crop or breed, of food production, of packaging including transport and distribution up to the point of sale. Secondly, it has to be checked, if the assumed coincidence of low energy turnover and regional origin can be verified. The specific energy turnover, calculated in the unit [kWh per kg] or [kWh per l] of food, is investigated by comparing regional with global process chains for different food items. Two examples of food – fruit juices and lamb meat – are researched, by personal investigation worldwide. Firstly, fruit juices of high grade quality from Brazil, from European origin and from local German farmers are compared, in terms of energy. Secondly, a comparison of lamb meat from New Zealand and lamb meat from local German farmers is conducted. Lamb meat has been investigated, because it is shipped around the world as frozen natural food, not concentrated like juices. In addition, the business size of the food producers is researched for both examples. Methods As a part of LCA the energy turnover of each process step from the very beginning up to the point of sale is investigated. These primary results are the basic empirical data, in order to allocate the energy turnover at the food items as functional units. The results of regional, European-continental and of global process chains are compared. In addition, the issue is investigated, whether the specific energy turnover depends upon the business size. Results and Discussion Surprisingly, the data in both cases demonstrate a strong degressive relation of the specific energy turnover and the business size. Here it is not important, if the business is regional or not. Merely the efficiency and logistics of the production and the operations determine the specific energy turnover. These findings seem closely connected with the business size, because small companies are not able to invest in energy recovering and saving technology. The regional juices business is worsened by the huge number of smallsized transports of the crop and the nearly bottle-wise distribution. Regarding lamb meat we once more find the disadvantages of comparably small farms again. In addition, the German farmers need daily shepherds, fences by night, stables and usually additional feed during the wintertime. All these efforts are not necessary in New Zealand, where the climatic conditions and the open countryside with low population allow rather easy and low energy breeding of lamb. Conclusion The coincidence of economic and ecological facts is obvious. As a matter of fact, in economics there is a strong degressive relation between the production costs and the number of produced items. This relation is very well known as “Economy of Scale.” Our findings lead to similar conclusions, in terms of ecology. That means, the production ecology depends on the number of produced items. Additionally, our results demonstrate a minimum business size, so that we can claim an “Ecology of Scale” as well. However, on the other hand, the reported data and the conclusions are valid for the investigated food items – fruit juices and lamb meat – only. Nevertheless, one conclusion is already evident: The most popular claims for regional food production and distribution instead of global process chains are not generally valid. Small farmers basically need much more energy to produce and distribute their products, compared with bigger units. Both food items demonstrate clearly, that the ecological quality is mainly influenced by the operational efficiency and not by the marketing distance itself. Recommendation and Outlook Much more detailed data of all the investigated operational units, processing fruit juices and lamb meat, have been published (Fleissner 2001). As a further example, we investigate wine from different countries as a further example. Different from fruit juices, which can be shipped as a concentrate, and different from lamb meat, which is shipped as frozen food, wine of high grade quality is always bottled close to the place of origin. That means, that not only the food, but also the heavy weight packaging is transported around the world. So, we are very curious about the results, which we expect for 2004.     

Client satisfaction with abortion care in three Russian cities

This paper explores client satisfaction with abortion care, looking at overall satisfaction and satisfaction with hygiene, comfort and courtesy. The analysis aimed to determine whether client satisfaction was related to the characteristics of the client, in order to guide quality improvement efforts. The analysis is a secondary analysis of data collected in a survey of 489 abortion clients who attended a limited number of hospitals in three cities in Russia. The data were analysed using logistic regression. Client characteristics, in general, did not affect overall satisfaction though there were significant differences in overall satisfaction for unmarried versus married women (OR=0.29, CI=0.13, 0.63). Similarly, most characteristics of the abortion visit were not related to client satisfaction, although women who were awake for the procedure were less likely to be satisfied (OR=0.37, CI=0.16, 0.89). Information provided to abortion clients about self-care was the most important predictor of overall satisfaction for abortion clients (OR=3.55, CI=1.64, 7.69). This suggests that improving the information provided to clients, through training providers and other staff, is important in these settings.     

Distribution of TRPC1 and TRPC5 in medial temporal lobe structures of mice

The transient receptor potential (TRP) superfamily comprises a group of non-selective cation channels that have been implicated in both receptor and store-operated channel functions. The family of the classical TRPs (TRPCs) consists of seven members (TRPC1-7). The presence of TRPC1 and TRPC5 mRNA in the brain has previously been demonstrated by real-time polymerase chain reaction. However, the distribution of these receptors within different brain areas of mice has not been investigated in detail. We have used antibodies directed against TRPC1 and TRPC5 to study the distribution and localization of these channels in murine medial temporal lobe structures. Both TRPC1 and TRPC5 channels are present in the various nuclei of the amygdala, in the hippocampus, and in the subiculum and the entorhinal cortex. We have found that TRPC1 channels are primarily expressed on cell somata and on dendrites, whereas TRPC5 channels are exclusively located on cell bodies. Moreover, TRPC1 channels are selectively expressed by neurons, whereas TRPC5 channels are mainly expressed by neurons, but also by non-neuronal cells. The expression of TRPC1 and TRPC5 channels in mammalian temporal lobe structures suggests their involvement in neuronal plasticity, learning and memory. This work was supported by the DFG (SFB 636/A5).