Diurnaler Säurerhythmus bei Tillandsia usneoides: Untersuchungen über den Weg des Kohlenstoffs sowie die Abhängigkeit des CO2-Gaswechsels von Lichtintensität,Temperatur und Wassergehalt der Pflanze

Summary Tillandsia usneoides, in the common sense a non-succulent plant, exhibits CO₂ exchange characterized by net CO₂ dark fixation during the night and depression of CO₂ exchange during the day. Malate has been demonstrated to accumulate during CO₂ dark fixation and to be converted to carbohydrates in light. Thus, T. usneoides exhibits CAM like typical succulents.Net CO₂ uptake during the day is increased with net CO₂ output being suppressed in duration of time and extent when light intensity increases. Furthermore, a slight increase in CO₂ fixation during the following night can be observed if the plants were treated with high light intensity during the previous day.Curves of CO₂ exchange typical for CAM are obtained if T. usneoides is kept at 15°C and 20°C. Lower temperature tend to increase CO₂ uptake during the day and to inhibit CO₂ dark fixation. Temperatures higher than 20°C favour loss of CO₂ by respiration, which becomes apparent during the whole day and night at 30°C and higher temperatures. Thus, T. usneoides gains carbon only at temperatures well below 25°C.Net CO₂ uptake during the day occurs only in moist plant material and is inhibited in plants cept under water stress conditions. However, CO₂ uptake during the night is clearly favoured if the plants dry out. Therefore dry plants gain more carbon than moist ones.Curves of CO₂ exchange typical for CAM were also obtained with 13 other species of the genus Tillandsia.The exhibition of CAM by the non-succulent T. usneoides calls for a new definition of the term succulence if it is to remain useful in characterizing this metabolic pathway. Because CO₂-fixing cells of T. usneoides possess relatively large vacuoles and are relatively poor in chloroplasts, they resembles the assimilatory cells of typical CAM-exhibiting succulents. Therefore, if succulence only means the capacity of big vacuoles to store malate, the assimilatory cells in T. usneoides are succulent. It seems to be useful to investigate parameters which would allow a definition of the term succulence on the level of the cell rather than on the level of the whole plant or plant organs.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Delayed and restricted expression limits putative instructional opportunities of Vgamma1.1/Vgamma2 gammadelta TCR in alphabeta/gammadelta lineage choice in the thymus

Development of alphabeta and gammadelta T cells depends on productive rearrangement of the appropriate TCR genes and their subsequent expression as proteins. TCRbeta and TCRgammadelta proteins first appear in DN3 and DN4 thymocytes, respectively. So far, it is not clear whether this is due to a delayed expression of TCRgammadelta proteins or to a more rapid progression to DN4 of thymocytes expressing TCRgammadelta. The answer to this question bears on the distinction between instructive and stochastic models of alphabeta/gammadelta lineage decision. To study this question, we first monitored initial TCR protein expression in wild-type and TCR transgenic mice in reaggregate thymic organ cultures. A TCRbeta transgene was expressed in nearly all DN3 and DN4 cells, accelerated DN3 to DN4 transition, and strongly diminished the number of cells that express TCRgammadelta proteins. In contrast, TCRgammadelta transgenes were expressed only in a fraction of DN4 cells, did not accelerate DN3 to DN4 transition, and did not reduce the number of DN4 cells expressing TCRbeta proteins. The TCRbeta transgene partially inhibited endogenous TCRgamma rearrangements, whereas the TCRgammadelta transgenes did not inhibit endogenous TCRbeta rearrangements. Second, we analyzed frequencies of productive TCRbeta and TCRgammadelta V(D)J junctions in DN3 and DN4 subsets. Most importantly, frequencies of productive TCRgammadelta rearrangements (Vdelta5, Vgamma1.1, and Vgamma2) appeared unselected in DN3. The results suggest a late and restricted expression of the corresponding gammadeltaTCR, severely limiting their putative instructional opportunities in alphabeta/gammadelta divergence.     

Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress

Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A₂ (hGX sPLA₂) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA₂ and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA₂ releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA₂ and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA₂ and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA₂-induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions.     

本溪地区蚊虫初步调查

本溪地区的蚊虫过去未有过文献报告。我们于1957—1958年,在本溪地区(本溪市、本溪县)进行了蚊虫的初步调查。经两年的调查已发现的蚊种,有按蚊属1种,伊蚊属4种,库蚊属6种,共计11种。其名称如下:     

Solution NMR Structure and Functional Analysis of the Integral Membrane Protein YgaP from Escherichia coli

The solution NMR structure of the α-helical integral membrane protein YgaP from Escherichia coli in mixed 1,2-diheptanoyl-sn-glycerol-3-phosphocholine/1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles is presented. In these micelles, YgaP forms a homodimer with the two transmembrane helices being the dimer interface, whereas the N-terminal cytoplasmic domain includes a rhodanese-fold in accordance to its sequence homology to the rhodanese family of sulfurtransferases. The enzymatic sulfur transfer activity of full-length YgaP as well as of the N-terminal rhodanese domain only was investigated performing a series of titrations with sodium thiosulfate and potassium cyanide monitored by NMR and EPR. The data indicate the thiosulfate concentration-dependent addition of several sulfur atoms to the catalytic Cys-63, which process can be reversed by the addition of potassium cyanide. The catalytic reaction induces thereby conformational changes within the rhodanese domain, as well as on the transmembrane α-helices of YgaP. These results provide insights into a potential mechanism of YgaP during the catalytic thiosulfate activity in vivo.     

Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [¹⁵N,¹H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [¹⁵N,¹H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.     

siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others’ supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2–8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.     

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.