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  1. Detailansicht für Treffer 1 öffnen
    Figure 10. LH neurons drive motor behaviours.(A) Schematic of the Flybowl optogenetic stimulation paradigm. ; Flies are introduced to the chamber and allowed to acclimatize for 30 s followed by illumination for 5 s, repeated three times. Each stimulation is of a different intensity, from lowest to highest. The flies were continuously tracked and their movements used to calculate the forward locomotion.The black bar above the 3rd stimulation represents the analysis window for D. (B) A graph of the forward locomotion (pixels per second, one pixel is ~0.12 mm) across time, where the split-GAL4 line LH1129 (magenta) is plotted against empty split-GAL4 (green). Line is the mean, grey is the SEM. Activation of LH1129, a split-GAL4 line labelling LH cell type AV6a1, drives an increase in forward locomotion compared to control. Red rectangle indicates light ON period of increasing intensity. (C) Z-projection of the brain expression patterns of the two split-GAL4 lines for cell type 7A, LH1139 (top) and LH1129 (bottom). Note minor trachea labelling in LH1129. All split-GAL4 lines are driving csChrimson::mVenus (green), the neuropil stain is nc82 (magenta). See Figure 10—figure supplement 1 for full brain and nerve cord expression patterns. (D) Boxplots analysing the mean locomotion during the strongest stimulation window of five seconds (locomotion measured in pixels per second, one pixel is ~0.12 mm). Each dot represents the behaviour of a single fly. Asterisk indicates significantly different from the empty split-GAL4 control. (E) Schematic of the flying optogenetic stimulation paradigm. Animals are tethered in a flight area. Flying animals have either no visual stimulus, a stationary bar or a closed loop blue bar. During flight optogenetic stimulation is driven by LED stimulation bilaterally. Schematic was modified with permission. (F–H) Z-projection of the brain expression patterns of the three split-GAL4 lines used in the flight assay: (F) LH304, (G) LH528 and (H) LH989. All split-GAL4 lines are driving csChrimson::mVenus (green). Note for all expression pattern images, the neuropil stain is nc82 (magenta). See Figure 10—figure supplement 1 for full brain and nerve cord expression patterns. (I) The absolute value of yaw (turning) over time (seconds) of single LH304 split-GAL4 flies crossed to experimental (magenta) and control genotypes (dark purple). This represents turning. (J) The wingbeat frequency over time (seconds) of single LH528 split-GAL4 flies crossed to experimental (magenta) and control genotypes (dark purple). The reduction in wingbeat frequency represents a reduction in speed and persists after stimulation. (K) The wingbeat thrust over time (seconds) of single LH989 split-GAL4 flies crossed to experimental (magenta) and control genotypes (dark purple). The increase in thrust represents increased power. WTB = Wild Type Berlin background strain. In I-K, lines are mean traces from all animals, coloured shades around the lines are standard errors of the mean across the animals (n = 6 for each genotype). The rectangle pink shades indicate the periods of activation light on. The red +marks above each plot indicate the time points at which the CsChrimson traces are significantly different from the negative control traces (Wilcoxon rank sum test, p
    2019
    unknown
    Wie komme ich dran?
  2. Detailansicht für Treffer 2 öffnen
    Figure 4. Dopamine metabolism is impaired in D. sechellia. ; (A) L-3,4-dihydroxyphenylalanine quantification (l-DOPA pg/fly) (N = 3) in whole fly, bodies and ovaries of female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd). *p < 0.05 and **p < 0.002 using Student's t test. (B) Relative L-3,4-dihydroxyphenylalanine (% pg l-DOPA per mg body, % l-DOPA) (N = 3) in female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd) or morinda diet (md). p = 0.0062 and p = 0.018 using Student's t test D. melanogaster vs D. sechellia fed, respectively, a standard diet or morinda diet. (C) Western blots of total protein whole-fly extracts for TH-PLE, CATSUP, and α-TUBULIN as a loading control, in D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd) or morinda diet (md). The numbers under TH-PLE and CATSUP protein lanes indicate the relative protein levels (normalised to α-TUBULIN). (D) Ratios of L-3,4-dihydroxyphenylalanine (l-DOPA/tyr) (N = 3) and dopamine (DA/tyr) (N = 3) to tyrosine substrate in female D. melanogaster wild-type Berlin (wtB) and D. sechellia (14021–0248.25, sec) fed a standard diet (sd). **p < 0.007 and ***p < 0.000007 using Student's t test. (E) Drosophila CATSUP protein structure scheme showing a signal peptide (grey box) and six trans-membrane domains (black boxes). Deletions (dash) and exchanges (grey or white) of amino acids in D. sechellia (14021–0248.25, sec) compared to in D. melanogaster wild-type Berlin (wtB) and in D. melanogaster DGRP-357 (DGRP-357) CATSUP are indicated. (F–H) Dopamine (DA ng/fly) (N = 3) (F), relative rate of vitellogenesis (% >S8/ 10) (G), and egg-volume (% mm3(10−3)) (n > 10) (H) in D. sechellia (14021–0248.25, sec), D. melanogaster wild-type Berlin (wtB) and D. melanogaster DGRP-357 (DGRP-357) fed a standard diet (sd). (I) Egg production (egg/female/day) (N > 3) in D. melanogaster wild-type Berlin (wtB), heterozygote flies (Catsup1/CyO), D. melanogaster DGRP-357 (CatsupIn270Del/CatsupIn270Del), trans heterozygote flies (CatsupIn270Del/Catsup1) and D. sechellia (14021–0248.25, sec), fed a standard diet (sd). Different letters denote significant differences (p < 0.05) using ANOVA followed by Tukey's test (F–I). Error bars represent s.e.m.
    2014
    unknown
    Wie komme ich dran?
  5. Detailansicht für Treffer 5 öffnen
    Figure 1. Morinda increases egg production in D. sechellia. ; (A–C) Egg production (egg/female/day) (N > 20) (A) and its relative change (N > 5) (B and C) in D. sechellia (14021–0248.25, sec) and D. melanogaster wild-type Berlin (wtB) fed a standard diet (sd), or morinda diet (md), or banana diet (bd), or diets supplemented with morinda carboxylic acids (+OA:HA). (D) Confocal images showing the surface (top) or the interior (middle) of ovarioles stained with nucleic acid specific dyes (sytox orange (SO) and TOTO) of D. sechellia (14021–0248.25) fed a standard diet (sec-sd) or a morinda diet (sec-md). The follicle cells (f) surrounding the oocyte (o) or stretched over the nurse cells (n) (arrow) are indicated for early (S8). Scale bar 100 μm. (E) Rate of vitellogenesis (>S8/ 8) in D. sechellia (14021–0248.25, sec) and D. melanogaster wild-type Berlin (wtB) fed a standard diet (sd), or a morinda diet (md), or a diet supplemented with morinda carboxylic acids (+OA:HA). Different letters denote significant differences (p < 0.01) using ANOVA followed by Tukey's test (B–E); ****p < 0.00001 using Student's t test to compare species (A). Error bars represent s.e.m.
    2014
    unknown
    Wie komme ich dran?
  6. Detailansicht für Treffer 6 öffnen
    Figure 2. Identification of pre-S1 binding protein on primary hepatocytes with photoreactive peptide Myr-47/WTb. ; (A) Left: Cultured PTHs at 24–48 hr after isolation and plating were photo-cross-linked with 200 nM Myr-47/WTb (WTb) or Myr-47/N9Kb (N9Kb), followed by Streptavidin Dynal T1 beads precipitation and Western blot analysis using mAb 2D3. The protein cross-linked by WTb is sensitive to PNGase F treatment and shifted from ∼65 to ∼43 kDa. Right: WTb cross-linked samples were treated with 100 mM DTT and/or PNGase F as indicated and detected similarly as in the left panel. (B) Non-photoreactive Myr-47/WT peptide (WT) but not its N9K mutant competed with 200 nM of WTb peptide for cross-linking with PTHs in a dose-dependent manner. (C) The abundance of the target protein(s) in PTH cells decreased over time. PTHs on different days of in vitro culturing were photo-cross-linked with 200 nM WTb. The cross-linked samples were analyzed by Western blot. The two bands at ∼65 and ∼43 kDa were due to incomplete deglycosylation by PNGase F. (D) WTb cross-linking with primary human hepatocytes (PHH). Frozen PHH cells were thawed and plated 1 day before cross-linking. With same procedure as in panel A, 200 nM WTb but not N9Kb cross-linked with a glycoprotein of molecular weight at ∼60 kDa, which shifted to ∼39 kDa upon PNGase F treatment. (E) Purification of target protein(s) for MS analysis. PTHs photo-cross-linked with 200 nM of WTb or N9Kb peptide were lysed, then the peptides and their cross-linked proteins were purified in tandem with Streptavidin Dynal T1 beads, mAb 2D3 conjugated beads, and Streptavidin Dynal T1 beads in 1× RIPA buffer. Extensive wash was applied for each purification step. The samples were treated with or without PNGase F as indicated prior to the last step of Streptavidin beads precipitation. The final purified samples were subjected to SDS-PAGE followed by silver staining (left). Bracketed areas indicate the bands cut for MS analysis. Western blot analysis (right) of the same cross-linked samples were performed similarly as in panel A. The top 10 nonredundant proteins identified in the 3 samples by MS analysis are listed in Figure 2—Source data 1. The common protein hit identified by MS analysis of the ∼65- and ∼43-kDa bands cut from the WTb cross-linked sample was Tupaia NTCP (tsNTCP), and the representative MS/MS spectra and parameters of the peptide hits are shown in Figure 2—figure supplement 5. The control band cut from N9Kb cross-linked sample did not generate any hits on any of these peptides. (F) Predicted tsNTCP protein sequence. A 30-amino acid insertion unique to tsNTCP is underlined. Two peptides identified by LC-MS/MS were highlighted in green. All lysine and arginine are highlighted in red to indicate trypsin cleavage sites. Many of the potential tryptic peptides are not appropriate for LC-MS detection because of unfavorable size and/or hydrophobicity. PTH: primary Tupaia hepatocytes; MS: mass spectrometry.
    2012
    unknown
    Wie komme ich dran?
  7. Detailansicht für Treffer 7 öffnen
    Figure 1—figure supplement 1. Morinda stimulates egg production in D. sechellia. ; Egg production (egg/female/day) in D. sechellia 14021–0248.25 (sec.25, N = 3), D. sechellia 14021–0248.31 (sec.31, N = 3), D. melanogaster wild-type Berlin (wtB, N = 3) and D. melanogaster Canton-S (CS, N = 3), fed a standard diet (sd) or morinda diet (md). ns = non-significant, *p < 0.05 using Student's t test to compare diets in each group. Error bars represent s.e.m.
    2014
    unknown
    Wie komme ich dran?
  11. Detailansicht für Treffer 11 öffnen
    Figure 4—figure supplement 2. Tyrosine quantification in Drosophila. ; D. sechellia (14021–0248.25) females (sec) show higher tyrosine content (expressed as picogram per fly [pg/fly]), compared to D. melanogaster wild-type Berlin females (wtB) and D. melanogaster DGRP-357 (DGRP-357) females, fed a standard diet. N = 3. Different letters denote significant differences (p < 0.05) using ANOVA followed by Tukey's test. Error bars represent s.e.m.
    2014
    unknown
    Wie komme ich dran?
  12. Detailansicht für Treffer 12 öffnen
    Figure 4—figure supplement 4. L-DOPA rescues diminished CatsupIn270Del/Catsup1 egg production. ; Relative egg production (% egg/female/day) (N = 3) in D. melanogaster wild type Berlin (wtB) and D. melanogaster trans heterozygote flies (CatsupIn270Del/Catsup1) fed a non-supplemented (−) standard diet or a diet supplemented with L-3,4-dihydroxyphenylalanine (1 mg/ml, l-DOPA). ns = non-significant; *p < 0.015 using Student's t test to compare diets in each group.
    2014
    unknown
    Wie komme ich dran?
  13. Detailansicht für Treffer 13 öffnen
    Figure 4—figure supplement 5. Conserved Catsup sequence in D. sechellia. ; Drosophila CATSUP amino acids sequence showing deletions (dash) and exchanges (grey or white) of amino acids in D. sechellia (14021–0248.03 (sec_03), 14021–0248.07 (sec_07), 14021–0248.08 (sec_08), 14021–0248.25 (sec_25), 14021–0248.28 (sec_28)) compared to in D. melanogaster wild-type Berlin (wtB).
    2014
    unknown
    Wie komme ich dran?
  14. Detailansicht für Treffer 14 öffnen
    Figure 2—figure supplement 5. Representative MS/MS spectra and parameters of the identified peptide hits. ; The (A) ∼65- and (B) ∼43-kDa bands cut from the WTb cross-linked sample (Figure 2E, left) were analyzed by mass spectrometry. The MS/MS spectra and parameters of the NTCP peptide hits are shown.
    2012
    unknown
    Wie komme ich dran?
  15. Detailansicht für Treffer 15 öffnen
    Figure 3. Binding of NTCP with N-terminal peptide of pre-S1 and HDV virions. ; (A) 293T cells transfected with an expression vector or plasmid containing cDNA of hNTCP or tsNTCP fused with a C9 tag at its C-terminus were cross-linked with 200 nM Myr-47/WTb or Myr-N9Kb similarly as in Figure 2A at 24 hr post-transfection. Cross-linked protein samples were precipitated by Streptavidin Dynal beads followed by treatment with PNGase F as indicated, and then analyzed by Western blotting using mAb 2D3 or anti-C9 tag antibody. (B) 293T cells transfected with tsNTCP-EGFP or a control hSDC2-EGFP (encoding human heparan sulfate proteoglycan core protein fused with EGFP at C-terminus) expression plasmid were incubated with WTb or N9Kb in the presence or absence of 200 nM non-photoreactive Myr-47/WT as indicated. Bound peptides were probed with PE-streptavidin and the colocalization of peptide and NTCP on cell surface was shown in the merged images. (C) FACS analysis of pre-S1 peptide binding with hNTCP transiently transfected Huh-7 cells. 24 hr post-transfection with hNTCP or a control plasmid, the cells were stained with 200 nM FITC-pre-S1 (FITC-labeled lipopeptide corresponding to the N-terminal 59-amino acid of pre-S1). The binding was analyzed by flow cytometry. (D) Huh-7 cells, after 24 hr of transfection of indicated plasmids, were incubated with wild-type HDV or HDV with a N9K mutation on its L protein. Bound virions were quantified by qRT-PCR. The result is presented as fold changes of binding over the background virus binding to pcDNA6-transfected cells. mAb: monoclonal antibody; tsNTCP: Tupaia NTCP; NTCP: sodium taurocholate cotransporting polypeptide.
    2012
    unknown
    Wie komme ich dran?
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