Notch1 control of oligodendrocyte differentiation in the spinal cord

We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.     

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

DNA damage formation and repair are tightly linked to protein–DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication (ARS1). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct interactions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.     

Investigations into the Genetic Variation, Population Structure, and Breeding Systems of the Fern Asplenium trichomanes subsp. quadrivalens

The genetic structure of five populations of the tetraploid fern Asplenium trichomanes subsp. quadrivalens in northern Switzerland was analyzed. Asplenium trichomanes subsp. quadrivalens is one of the most common and most widespread ferns in Europe. In this study we have combined genetic investigations, spatial autocorrelation, and breeding experiments to investigate in detail five populations from natural rock faces. Enzyme electrophoresis revealed very low genetic variability within and among the populations. The small amount of variation was partitioned mainly among the localities, indicated by high Fst values up to 0.764. Overall means of the proportion of polymorphic loci (P=0.076), the mean number of alleles per locus (A=1.086), and the mean expected heterozygosity (H=0.018) were low compared with other ferns (e.g., Kirkpatrick et al. 1990). Very few heterozygous individuals were found. Values of the fixation index (F) were high, ranging between 0.732 and 1.000 and indicating substantial inbreeding. Spatial autocorrelation showed different patterns of substructure in populations of A. trichomanes subsp. quadrivalens with a tendency for patches in short distances (up to 1.5 m). The breeding experiments with isolated prothalli and prothalli pairs showed that a mean of 56.4% of the isolated prothalli were successful in sporophyte formation. The highest rate in one population was 83.3%. We conclude that genetic load must be low in A. trichomanes subsp. quadrivalens. Sporophyte formation was statistically more successful in the experiments with gametophyte pairs than in isolates, indicating that additional cross-fertilization occurred. The latter agreed with the occurrence of few heterozygote samples and the small number of multilocus phenotypes found in natural habitats. Asplenium trichomanes subsp. quadrivalens is shown to be a highly inbreeding taxon with the capability of single spore colonization and subsequent founding of new populations. Such features can be hypothesized to have contributed to the postglacial colonization and the widespread distribution of this taxon in Europe.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.     

Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay)

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.     

Nitrogen deposition but not ozone affects productivity and community composition of subalpine grassland after 3 yr of treatment

A field experiment was established at 2000 m above sea level (asl) in the central Swiss Alps with the aim of investigating the effects of elevated ozone (O(3)) and nitrogen deposition (N), and of their combination, on above-ground productivity and species composition of subalpine grassland. One hundred and eighty monoliths were extracted from a species-rich Geo-Montani-Nardetum pasture and exposed in a free-air O(3)-fumigation system to one of three concentrations of O(3) (ambient, 1.2 x ambient, 1.6 x ambient) and five concentrations of additional N. Above-ground biomass, proportion of functional groups and normalized difference vegetation index (NDVI) were measured annually. After 3 yr of treatment, the vegetation responded to the N input with an increase in above-ground productivity and altered species composition, but without changes resulting from elevated O(3). N input > 10 kg N ha(-1) yr(-1) was sufficient to affect the composition of functional groups, with sedges benefiting over-proportionally. No interaction of O(3) x N was observed, except for NDVI; positive effects of N addition on canopy greenness were counteracted by accelerated leaf senescence in the highest O(3) treatment. The results suggest that effects of elevated O(3) on the productivity and floristic composition of subalpine grassland may develop slowly, regardless of the sensitive response to increasing N.     

Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.