Ddx42p--a human DEAD box protein with RNA chaperone activities

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)⁺ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.     

Compensation of large motion sensor displacements during long recordings of limb movements

In motion capture applications using electromagnetic tracking systems the process of anatomical calibration associates the technical frames of sensors attached to the skin with the human anatomy. Joint centers and axes are determined relative to these frames. A change of orientation of the sensor relative to the skin renders this calibration faulty. This sensitivity regarding sensor displacement can turn out to be a serious problem with movement recordings of several minutes duration. We propose the “dislocation distance” as a novel method to quantify sensor displacement and to detect gradual and sudden changes of sensor orientation. Furthermore a method to define a so called fixed technical frame is proposed as a robust reference frame which can adapt to a new sensor orientation on the skin. The proposed methods are applied to quantify the effects of sensor displacement of 120 upper and lower limb movement recordings of newborns revealing the need for a method to compensate for sensor displacement. The reliability of the fixed technical frame is quantified and it is shown that trend and dispersion of the dislocation distance can be significantly reduced. A working example illustrates the consequences of sensor displacement on derived angle time series and how they are avoided using the fixed technical frame.     

Subcellular volumes and metabolite concentrations in barley leaves

Metabolite concentrations in subcellular compartments from mature barley (Hordeum vulgare L. cv. Apex) leaves after 9 h of illumination and 5 h of darkness were determined by nonaqueous fractionation and by the stereological evaluation of cellular and subcellular volumes from light and electron micrographs. Twenty one-day-old primary leaves of barley with a total leaf volume of 902 μL per mg chlorophyll were found to be composed of 27% epidermis, 42% mesophyll cells, 6% veins, 4.5% apoplast and 23% gas space. While in epidermal cells 99% of the volume was occupied by the vacuole, mesophyll cells with an average volume of 31.3 pL consisted of 23 pL (73%) vacuole, 4.6 pL (19%) chloroplasts, 2.06 pL (6,7%) cytosol (including smaller organelles and vesicles), 0.34 pL (1%) mitochondria and 107 fL (0.34%) nucleus. The differences between leaves harvested after 9 h of illumination and after 5 h of darkness were in the size of the stromal compartment and the starch grains therein. Subcellular metabolite concentrations were calculated from the compartmental volumes and metabolite contents of the compartments as determined by nonaqueous fractionation. The amino-acid concentrations in stroma and cytosol were rather similar after 9 h of illumination and 5 h of darkness. In contrast, the vacuolar amino-acid concentrations were about one order of magnitude lower than the stroma and cytosol values, and there was a slight increase in concentration after 5 h of darkness.     

Ala31-Aib32: identification of the key motif for high affinity and selectivity of neuropeptide Y at the Y5-receptor

The turn-inducing sequence Ala-Aib introduced into positions 31 and 32 of neuropeptide Y (NPY) and its analogues has been identified as the key structure for Y(5)-receptor selectivity. Analogues of NPY and PP/NPY chimera containing the motif Ala-Aib were prepared; these peptides turned out to be selective for the Y(5)-receptor. The affinity of the NPY-based peptides was in the range of 6-150 nM, while the affinity of three (Ala-Aib)-containing PP/NPY chimera was in the range of 0.2-0.9 nM. The circular dichroism spectra of the Aib analogues in aqueous solution were all characteristic of an alpha helix; however, they had different intensities of the two negative bands at 220 and 208 nm. Affinity and selectivity for the Y(5)-receptor were correlated with the ratio of the ellipticity at 220 nm versus the one at 208 nm (R), which indicates the presence of a pronounced helix (R > 1) versus a less stabile one (R < 1). When R was in the range 0.74-0.96, the affinity at the Y(5)-receptor was in the range >5 nM, while there was complete loss of affinity at the Y(4)-receptor. R > 1.15 was associated with very high affinity at the Y(5)-receptor and weak affinity at the Y(4)-receptor. These results suggest that the selectivity of the Ala(31)-Aib(32) motif for the Y(5)-receptor derives from a specific conformation that must be correlated with the bioactive conformation of NPY at this subtype.     

Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors

Local excess of nitric oxide (NO) has been implicated in beta-cell damage, thus, a possible approach to the treatment of autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahydropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently evaluated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells and a combination of IL1-1 beta and IFN-gamma as inducers of cellular NO production. The structure-activity analysis revealed that the variation of substituents in the position 1 of the hexahydropyridazine strongly influences the inhibitory activity to iNOS as well as being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly significant inhibitory effects. However, for an efficient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxyphenyl, and 4-methoxyphenyl (IC(50): 0.5-2.1 mM), respectively, provided compounds with similar inhibitory activity to aminoguanidine (IC(50): 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides, which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously substituted aromatic rings.     

Co-chaperone BAG3 enters autophagic pathway via its interaction with microtubule associated protein 1 light chain 3 beta

The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with chaperones of the HSP70 family or small HSPs. During aging and under cellular stress conditions in particular, BAG3, together with molecular chaperones, ensures the sequestration of aggregated or aggregation-prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. Accumulating evidence for BAG3-mediated selective autophagy independent of cargo ubiquitination led to analyses predicting a direct interaction of BAG3 with LC3 proteins. Phylogenetically, BAG3 comprises several highly conserved potential LIRs, LC3-interacting regions, which might allow for the direct targeting of BAG3 including its cargo to autophagosomes and drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with LC3B. In addition, we could demonstrate that disabling all predicted LIRs abolished the inducibility of a colocalization of BAG3 with LC3B-positive structures and resulted in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared with wild-type BAG3. These results suggest an autophagic targeting of BAG3 via interaction with LC3B. Therefore, we conclude that, in addition to being a key co-chaperone to HSP70, BAG3 may also act as a cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control.     

The German wildlife information system: population densities and development of European Hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis> PALLAS) during 2002–2005 in Germany

The German Wildlife Information System, founded in 2001, is a long-term monitoring program documenting occurrence, number, and development of game populations throughout Germany. Population numbers are recorded by standardized counting methods in so-called reference areas. The population densities of the European hare are calculated by spotlight strip censuses in the reference areas each spring and autumn all across Germany. From 2002 to 2005, the censuses were carried out by local hunters in 510 to 676 reference areas each year. During these years, the calculated spring densities increased significantly from 11.0 (2002) to 14.5 hares/km² (2005) nationwide. The overall increase in spring densities was primarily caused by the population rise from spring 2003 to 2004, which correlates with the high net growth rate in 2003. In 2005, the number of counted hares varied between less than 1 and more than 107 hares/km² in spring and between 0 and more than 170 hares/km² in autumn. Because of differing landscapes in Germany, three regions were differentiated. In spring 2005, the average population densities (median) in East Germany (5.4 hares/km²) and Southwest Germany (14.6 hares/km²) were significantly lower than in Northwest Germany (23.9 hares/km²). These regional differences had been similarly distinct in former years.