Conditional MLL-CBP targets GMP and models therapy-related myeloproliferative disease

Chromosomal translocations that fuse the mixed lineage leukemia (MLL) gene with multiple partners typify acute leukemias of infancy as well as therapy-related leukemias. We utilized a conditional knockin strategy to bypass the embryonic lethality caused by MLL-CBP expression and to assess the immediate effects of induced MLL-CBP expression on hematopoiesis. Within days of activating MLL-CBP, the fusion protein selectively expanded granulocyte/macrophage progenitors (GMP) and enhanced their self-renewal/proliferation. MLL-CBP altered the gene expression program of GMP, upregulating a subset of genes including Hox a9. Inhibition of Hox a9 expression by RNA interference demonstrated that MLL-CBP required Hox a9 for its enhanced cell expansion. Following exposure to sublethal gamma-irradiation or N-ethyl-N-nitrosourea (ENU), MLL-CBP mice developed myelomonocytic hyperplasia and progressed to fatal myeloproliferative disorders. These represented the spectrum of therapy-induced acute myelomonocytic leukemia/chronic myelomonocytic leukemia/myelodysplastic/myeloproliferative disorder similar to that seen in humans possessing the t(11;16). This model of MLL-CBP therapy-related myeloproliferative disease demonstrates the selectivity of this MLL fusion for GMP cells and its ability to initiate leukemogenesis in conjunction with cooperating mutations.     

Human endo-alpha1,2-mannosidase is a Golgi-resident type II membrane protein

The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus.     

Bacteria and mucosal inflammation of the gut: lessons from Helicobacter pylori

The human gastrointestinal tract is colonized by an abundance of bacteria, which are in constant interaction with the epithelial lining usually leading to an intricate balance between tolerance and immunological response. There is ample evidence that the abundant presence of bacteria thus plays a role in the maintenance of human health, as well as in the induction of chronic inflammatory diseases of the gastrointestinal tract. Research in this field is, however, considerably hampered by the abundance of bacterial species, many of which have not even been characterized, and are difficult to culture specifically. These important limitations may to some extent be overcome by recent molecular biologic methods. Furthermore however, the adherent mucosal flora may differ largely from the luminal flora and that in excreta. These characteristics do not pertain to Helicobacter pylori, which generally colonizes the human stomach as a single strain with stable characteristics. Such colonization is stable throughout life, but can be treated. Furthermore, the association with chronic gastritis is very strong. For these reasons, H. pylori serves as an excellent model for the understanding of the processes involved in bacterial colonization and host response including mediation of immunoregulation, and the mechanisms by which this response can lead to disease.     

Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.     

The plant's capacity in regulating resource demand

Regulation of resource allocation in plants is the key to integrate understanding of metabolism and resource flux across the whole plant. The challenge is to understand trade-offs as plants balance allocation between different and conflicting demands, e.g., for staying competitive with neighbours and ensuring defence against parasites. Related hypothesis evaluation can, however, produce equivocal results. Overcoming deficits in understanding underlying mechanisms is achieved through integrated experimentation and modelling the various spatio-temporal scaling levels, from genetic control and cell metabolism towards resource flux at the stand level. An integrated, interdisciplinary research concept on herbaceous and woody plants and its outcome to date are used, while drawing attention to currently available knowledge. This assessment is based on resource allocation as driven through plant-pathogen and plant-mycorrhizosphere interaction, as well as competition with neighbouring plants in stands, conceiving such biotic interactions as a "unity" in the control of allocation. Biotic interaction may diminish or foster effects of abiotic stress on allocation, as changes in allocation do not necessarily result from metabolic re-adjustment but may obey allometric rules during ontogeny. Focus is required on host-pathogen interaction under variable resource supply and disturbance, including effects of competition and mycorrhization. Cost/benefit relationships in balancing resource investments versus gains turned out to be fundamental in quantifying competitiveness when related to the space, which is subject to competitive resource exploitation. A space-related view of defence as a form of prevention of decline in competitiveness may promote conversion of resource turnover across the different kinds of biotic interaction, given their capacity in jointly controlling whole plant resource allocation.     

Beech leaf colonization by the endophyte Apiognomonia errabunda dramatically depends on light exposure and climatic conditions

Ozone and light effects on endophytic colonization by Apiognomonia errabunda of adult beech trees (Fagus sylvatica) and their putative mediation by internal defence compounds were studied at the Kranzberg Forest free-air ozone fumigation site. A. errabunda colonization was quantified by "real-time PCR" (QPCR). A. errabunda-specific primers allowed detection without interference by DNA from European beech and several species of common genera of plant pathogenic fungi, such as Mycosphaerella, Alternaria, Botrytis, and Fusarium. Colonization levels of sun and shade leaves of European beech trees exposed either to ambient or twice ambient ozone regimes were determined. Colonization was significantly higher in shade compared to sun leaves. Ozone exhibited a marginally inhibitory effect on fungal colonization only in young leaves in 2002. The hot and dry summer of 2003 reduced fungal colonization dramatically, being more pronounced than ozone treatment or sun exposure. Levels of soluble and cell wall-bound phenolic compounds were approximately twice as high in sun than in shade leaves. Acylated flavonol 3- O-glycosides with putatively high UV-B shielding effect were very low in shade canopy leaves. Ozone had only a minor influence on secondary metabolites in sun leaves. It slightly increased kaempferol 3- O-glucoside levels exclusively in shade leaves. The frequently prominent hydroxycinnamic acid derivative, chlorogenic acid, was tested for its growth inhibiting activity against Apiognomonia and showed an IC50 of approximately 8 mM. Appearance of Apiognomonia-related necroses strongly correlated with the occurrence of the stress metabolite, 3,3',4,4'-tetramethoxybiphenyl. Infection success of Apiognomonia was highly dependent on light exposure, presumably affected by the endogenous levels of constitutive phenolic compounds. Ozone exerted only minor modulating effects, whereas climatic factors, such as pronounced heat periods and drought, were dramatically overriding.     

Characterization of susceptibility and resistance responses to potato cyst nematode (Globodera spp.) infection of tomato lines in the absence and presence of the broad-spectrum nematode resistance Hero gene

The tomato Hero A gene is the only member of a multigene family that confers a high level (>80%) of resistance to all the economically important pathotypes of potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida. Although the resistance levels of transgenic tomato lines were similar to those of the tomato line LA1792 containing the introgressed Hero multigene family, transgenic potato plants expressing the tomato Hero A gene are not resistant to PCNs. Comparative microscopy studies of in vitro infected roots of PCN-susceptible tomato cv. Money Maker, the resistant breeding line LA1792, and transgenic line L10 with Ro1 pathotype have revealed no statistically significant difference in the number of juveniles invading roots. However, syncytia (specialized feeding cells) induced in LA1792 and L10 roots mostly were found to have degenerated a few days after their induction, and a few surviving syncytia were able to support only the development of males rather than females. Thus, the ratio between males and females was biased towards males on LA1792 and L10 roots. A series of changes occur in resistant plants leading to formation of a layer of necrotic cells separating the syncytium from stellar conductive tissues and this is followed by degradation of the syncytium. Although the Hero A gene is expressed in all tissues, including roots, stems, leaves, and flower buds, its expression is upregulated in roots in response to PCN infection. Moreover, the expression profiles of the Hero A correlates with the timing of death of the syncytium.     

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis . In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) , which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress . Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. Here we provide a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired . Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. We propose a model of eIF4E regulation that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2.