Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli.We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.     

Rasl11b knock down in zebrafish suppresses one-eyed-pinhead mutant phenotype

The EGF-CFC factor Oep/Cripto1/Frl1 has been implicated in embryogenesis and several human cancers. During vertebrate development, Oep/Cripto1/Frl1 has been shown to act as an essential coreceptor in the TGFbeta/Nodal pathway, which is crucial for germ layer formation. Although studies in cell cultures suggest that Oep/Cripto1/Frl1 is also implicated in other pathways, in vivo it is solely regarded as a Nodal coreceptor. We have found that Rasl11b, a small GTPase belonging to a Ras subfamily of putative tumor suppressor genes, modulates Oep function in zebrafish independently of the Nodal pathway. rasl11b down regulation partially rescues endodermal and prechordal plate defects of zygotic oep(-/-) mutants (Zoep). Rasl11b inhibitory action was only observed in oep-deficient backgrounds, suggesting that normal oep expression prevents Rasl11b function. Surprisingly, rasl11b down regulation does not rescue mesendodermal defects in other Nodal pathway mutants, nor does it influence the phosphorylation state of the downstream effector Smad2. Thus, Rasl11b modifies the effect of Oep on mesendoderm development independently of the main known Oep output: the Nodal signaling pathway. This data suggests a new branch of Oep signaling that has implications for germ layer development, as well as for studies of Oep/Frl1/Cripto1 dysfunction, such as that found in tumors.     

Nanosecond pulsed electric fields cause melanomas to self-destruct

We have discovered a new, drug-free therapy for treating solid skin tumors. Pulsed electric fields greater than 20 kV/cm with rise times of 30 ns and durations of 300 ns penetrate into the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop. Melanomas shrink by 90% within two weeks following a cumulative field exposure time of 120 micros. A second treatment at this time can result in complete remission. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin. Each pulse deposits 0.2 J and 100 pulses increase the temperature of the treated region by only 3 degrees C, ten degrees lower than the minimum temperature for hyperthermia effects.     

An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.     

Assessment of the eutrophication status of transitional,coastal and marine waters within OSPAR

Eutrophication (nutrient enrichment and subsequent processes) and its adverse ecosystem effects have been discussed as main issues over the last 20 years in international conferences and conventions for the protection of the marine environment such as the North Sea Conferences and the 1992 OSPAR Convention (OSPAR; which combined and updated the 1972 Oslo Convention on dumping waste at the sea and the 1974 Paris Convention on land-based sources of marine pollution). OSPAR committed itself to reduce phosphorus and nitrogen inputs (in the order of 50% compared with 1985) into the marine areas and ‘to combat eutrophication to achieve, by the year 2010, a healthy marine environment where eutrophication does not occur’. Within OSPAR, the Comprehensive Procedure (COMPP) has been developed and used to assess the eutrophication status of the OSPAR maritime area in an harmonised way. This is based on classification in terms of the following types of areas Non-Problem Areas (no effects), Potential Problem Areas (not enough data to assess effects) and Problem Areas (effects due to elevated nutrients and/or due to transboundary transport from adjacent areas). The COMPP consists of a set of harmonised assessment criteria with their area-specific assessment levels and an integrated area classification approach. The criteria cover all aspects of nutrient enrichment (nutrient inputs, concentrations and ratios) as well as possible direct effects (e.g. increased levels of nuisance and/or toxic phytoplankton species, shifts and/or losses of submerged aquatic vegetation) and indirect effects (e.g. oxygen deficiency, changes and/or death of benthos, death of fish, algal toxins). The COMPP also includes supporting environmental factors. It takes account of synergies and harmonisation with the EC Water Framework Directive, and has formed a major basis for the EC eutrophication guidance. Recently, additional components, such as total nitrogen, total phosphorus and transboundary transports have been included in the assessment of, e.g. the German Bight. The second application of the COMPP resulting in an update of the eutrophication status of the OSPAR maritime area will be finalised in 2008, and will include the agreed integrated set of Ecological Quality Objectives (EcoQOs) with respect to eutrophication. Guest editors: J. H. Andersen & D. J. Conley Eutrophication in Coastal Ecosystems: Selected papers from the Second International Symposium on Research and Management of Eutrophication in Coastal Ecosystems, 20–23 June 2006, Nyborg, Denmark     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.