Colon cancer metastasis: MACC1 and Met as metastatic pacemakers

Colon cancer is still the second most frequent malignancy in the Western world. Despite major efforts in diagnosis and treatment it is one of the leading causes of cancer related deaths. The metastatic dissemination of primary tumors is directly linked to patient's survival and accounts for about 90% of all colon cancer deaths. Current clinical predictions on whether colon cancer will metastasize are mainly defined by histopathological staging, describing the tumor spread within a surgical specimen. This review focuses on the need for molecule-based staging as essential prerequisite for individualized diagnosis, prognosis and therapy. Molecular determinants for progression and metastasis of colon cancer are discussed. Moreover, a newly identified molecule playing a decisive role in colon cancer metastasis is highlighted: MACC1. MACC1 acts as a key regulator of the metastasis-inducing HGF/Met pathway, predicts the risk for metastasis in early cancer stages, and represents a novel target to attack metastasis.     

Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation

Wnt signalling plays important roles in patterning and outgrowth of the vertebrate limb. Different mutations in Wnt genes, their antagonists or (co-)receptors result in patterning and outgrowth defects as well as chondrocyte and bone phenotypes in mouse and human. Understanding Wnt activity during mouse limb development and chondrogenesis requires a temporal and spatial overview of Wnt signalling key factor expression. Here we present a comparative expression analysis of all 19 Wnt genes and their major secreted antagonists of the Dickkopf (Dkk), Wisp and the secreted frizzled related protein (Sfrp) families during mouse limb development. Our study reveals new domains of expression for Wnt2, Wnt2b, Wnt5b, Wnt6, Wnt7b, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16, in the limb. We also identified novel expression domains for the Wnt antagonists Sfrp1, Sfrp3, Sfrp5, Wisp1 as well as Dkk2 and Dkk3. We provide a full expression pattern for Wif1 in limb development, for which no limb expression had been documented so far.     

Distinct ex vivo susceptibility of B-cell subsets to epstein-barr virus infection according to differentiation status and tissue origin

Epstein-Barr virus (EBV) uses tonsils as the portal of entry to establish persistent infection. EBV is found in various B-cell subsets in tonsils but exclusively in memory B cells in peripheral blood. The in vitro susceptibilities of B-cell subsets to EBV infection have been studied solely qualitatively. In this work, we examined quantitatively the in vitro susceptibilities of various B-cell subsets from different tissue origins to EBV infection. First, we established a centrifugation-based inoculation protocol (spinoculation) that resulted in a significantly increased proportion of infected cells compared to that obtained by conventional inoculation, enabling a detailed susceptibility analysis. Importantly, B-cell infection occurred via the known EBV receptors and infected cells showed EBV mRNA expression patterns similar to those observed after conventional inoculation, validating our approach. Tonsillar na?ve and memory B cells were infected ex vivo at similar frequencies. In contrast, memory B cells from blood, which represent B cells from various lymphoid tissues, were infected at lower frequencies than their na?ve counterparts. Immunoglobulin A (IgA)-positive or IgG-positive tonsillar memory B cells were significantly more susceptible to EBV infection than IgM-positive counterparts. Memory B cells were transformed with lower efficiency than na?ve B cells. This result was paralleled by lower proliferation rates. In summary, these data suggest that EBV exploits the B-cell differentiation status and tissue origin to establish persistent infection.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

Formation of phosphatidic acid in stressed mitochondria

Mitochondria are an important intracellular source of ROS as well as a sensitive target for oxidative damage under certain pathological conditions such as iron or copper overload. Mitochondrial membranes are rich in the tetraacyl phospholipid cardiolipin. Its integrity is important for efficient oxidative phosphorylation. Mouse liver mitochondria were subjected to oxidative stress by the Cu²⁺(Fe²⁺)/H₂O₂/ascorbate system. Phosphatidic acid was detected in oxidized mitochondria, but not in unperturbed mitochondria. The Cu²⁺/H₂O₂/and (or not) ascorbate system caused the formation of phosphatidic acid and phosphatidylhydroxyacetone in cardiolipin liposomes. These products proceed via an HO-radical induced fragmentation taking place in the polar moiety of cardiolipin. Mass spectrometry analysis of phosphatidic acid newly formed in mitochondria revealed that it has been derived from fragmentation of cardiolipin. Thus, free-radical fragmentation of cardiolipin in its polar part with the formation of phosphatidic acid is a likely mechanism that damages mitochondria under conditions of oxidative stress.     

Sequence of the 50-kb Conjugative Multiresistance Plasmid pRE25 from Enterococcus faecalis RE25

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Identification and characterization of small RNAs from the phloem of Brassica napus

Systemic signalling is indispensable for the coordination of diverse physiological processes during development, defence and nutrient allocation. Indirect evidence suggests that plant small RNAs (smRNAs) could be involved in long-distance information transfer via the vasculature of the plant. Analyses of the smRNA complements of vascular exudates from oilseed rape ( Brassica napus ) showed that xylem sap is devoid of RNA, whereas phloem sap contained a large number of smRNAs. In addition to 32 annotated microRNAs (miRNAs) from 18 different families that could be identified and approved, a set of unknown smRNAs, predominantly of 21 and 24 nucleotides in length, was obtained, and selected candidates were found to be highly abundant in phloem sap. Moreover, we could demonstrate that the levels of three miRNAs known to respond to nutrient deprivation in non-vascular tissue, miR395 (sulphate), miR398 (copper) and miR399 (phosphate), were increased in phloem sap during the growth of plants under the respective starvation conditions. Interestingly, only mature miRNA molecules were found to be stress responsive, demonstrating that single-stranded sense miRNAs are most likely to represent the physiologically relevant molecules. The strong responses in the phloem suggest a role of miRNAs in systemic information transfer via this long-distance transport system.