Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Yields and nitrogen nutrition of intercropped maize and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi)

Maize (Zea mays L.) and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi) were grown in intercrop and monoculture on Tropaqualf soils under rainfed conditions in Northern Thailand yearly from 1983 to 1986. De Wit's replacement design was used to compare intercrops and monocultures with a constant plant density equivalent to 80 000 maize or 160 000 ricebean plants ha⁻¹. Combined nitrogen was applied at varying levels to 200 kg N ha⁻¹. In the final two seasons the intercrop ratio of maize: ricebean was also varied. At the time of maize maturity intercrops yielded upt 49 kg ha⁻¹ more N in the above ground plant parts than the best monoculture. Dry matter, grain and nitrogen yield of maize and ricebean in intercrop relative to their monoculture yields (RY, relative yield) were significantly greater than their respective share of the plant population. Relative yield totals (RYT) for grain, dry matter and nitrogen were always greater than 1. Nitrogen uptake per maize plant increased with progressive replacement of maize by ricebean plants. This increase was similar to that obtained by applying combined N. Available soil nitrogen tended to decrease with increasing maize:ricebean ratio. Increasing the maize:ricebean ratio increased the % of nitrogen derived from fixation in ricebean, the increase being equivalent to that obtained by decreasing combined nitrogen application. Approximately the same amount of fertilizer and soil nitrogen was taken up by maize plus ricebean in intercrop as the maize monoculture. The results suggest that the improved nitrogen economy of the intercrop resulted from the strong competitiveness of maize in the use of mineral nitrogen and the enhancement of nitrogen fixation in intercropped ricebean which made it less dependent on the depleted pool of soil nitrogen.     

USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Marine moult migration of the freshwater Scaly‐sided Merganser Mergus squamatus revealed by stable isotopes and geolocators

Scaly‐sided Mergansers Mergus squamatus breed on freshwater rivers in far eastern Russia, Korea and China, wintering in similar habitats in China and Korea, but nothing was known of their moulting habitat. To investigate the moult strategies of this species, we combined wing feather stable isotope ratios (males and females) with geolocator data (nesting females) to establish major habitat types (freshwater, brackish or saltwater) used by both sexes during wing moult. Although most Scaly‐sided Mergansers of both sexes probably moult on freshwater, some males and non‐breeding and failed breeding females appeared to undertake moult migration to brackish and marine waters. Given the previous lack of any surveys of coastal or estuarine waters for this species during the moult period, these findings suggest important survey needs for the effective conservation of the species during the flightless moult period.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.