Candidate amino acids involved in H+ gating of acid-sensing ion channel 1a

Acid-sensing ion channels are ligand-gated cation channels, gated by extracellular H+. H+ is the simplest ligand possible, and whereas for larger ligands that gate ion channels complex binding sites in the three-dimensional structure of the proteins have to be assumed, H+ could in principle gate a channel by titration of a single amino acid. Experimental evidence suggests a more complex situation, however. For example, it has been shown that extracellular Ca2+ ions compete with H+; probably Ca2+ ions bound to the extracellular loop of ASICs stabilize the closed state of the channel and have to be displaced before the channel can open. In such a scheme, amino acids contributing to Ca2+ binding would also be candidates contributing to H+ gating. In this study we systematically screened more than 40 conserved, charged amino acids in the extracellular region of ASIC1a for a possible contribution to H+ gating. We identified four amino acids where substitution strongly affects H+ gating: Glu63, His72/His73, and Asp78. These amino acids are highly conserved among H+-sensitive ASICs and are candidates for the “H+ sensor” of ASICs

Horstsaat von Buschbohnen bietet Möglichkeit zu ‚InRow‘-Hackmaßnahmen

Am Sächsischen Landesamt für Umwelt, Landwirtschaft und Geologie in Dresden-Pillnitz wurde 2017 die Horstsaat von Buschbohnen in Hinblick auf ihr Potential von ‚InRow‘-Hackmaßnahmen untersucht. Bei relativ einheitlicher Bestandesdichte von 36 Pfl./m² zeigten Horstsaatvarianten mit Ablage von 5 oder 7 Korn/Horst bzw. 26,0/36,4 cm Horstabstand maximal 10 % niedrigere Erträge als eine standardmäßige Einzelkornsaat mit 5,2 cm Kornablageabstand. Eine Beeinträchtigung der maschinellen Pflückbarkeit bei Horstsaat konnte nicht festgestellt werden. Bei Horstsaat konnten bis zu 67 % des Reihenbereichs gehackt werden, was sich in einer entsprechenden Reduzierung des Jätaufwandes widerspiegelte

Tfg (Trk fused gene) is a Carma-1/IKKgamma interacting protein involved in CD40-induced canonical NF-KB signaling

Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-betaB. In B cells, Carma-1 becomes phosphorylated by PKCbeta, leading to its oligomerization. Subsequent Bcl10 binding induces IKKbeta-activation and, thereby, canonical NF-KB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKgamma and to enhance TNFinduced NF-KB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKgamma and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7–1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKgamma complexes, fused IKKgamma, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-KB and IKKbeta. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKbeta activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-KB signals

IN VITRO INDUCTION OF TUMOR-SPECIFIC IMMUNITY : I. PARAMETERS OF ACTIVATION AND CYTOTOXIC REACTIVITY OF MOUSE LYMPHOID CELLS IMMUNIZED IN VITRO AGAINST SYNGENEIC AND ALLOGENEIC PLASMA CELL TUMORS

Induction of tumor-specific immunity in vitro was accomplished by cocultivation of cortisone-resistant murine thymocytes or spleen cells with irradiated syngeneic plasma cell tumors (PCT). The cytotoxic activity generated could be detected in a short-term 51Cr-release assay. Optimal cytotoxic activity against PCT-associated transplantation antigens (TATA) was generated after 7 days in culture. Unlike cytotoxic responses to tumor allografts in which the cytotoxic activity was directed against allogeneic transplantation antigens, the cytotoxic activity obtained in the syngeneic tumor system was specific to the immunizing syngeneic PCT. Similar parameters of induction of cytotoxic responses in in vitro tumor allograft responses and in the syngeneic tumor system suggested that both reactions are cell-mediated cytotoxic immune responses. With regard to the magnitude of cytotoxic responses obtained, allogeneic transplantation antigens induced about a 30-fold higher cytotoxic immune response than plasma cell TATA. The results are consistent with the concept that in vitro tumor allograft responses and in vitro responses against TATA of PCT are similar in quality, but differ in the magnitude of the cytotoxic response provoked

Prerequisites of High Resolution Scanning Electron Microscopy

Cryotechniques must be employed throughout all preparation and observation steps in order to extract high resolution scanning electron microscopical information from biological material. Cryoimmobilization, followed by freeze-drying and metal-shadowing at low temperature, yields optimal structural information of T4 polyheads used as a test specimen. Freeze-substitution of frozen T4 polyheads and subsequent freeze-drying renders the substructures recognizable but less crisp than freeze-drying from aequous solutions. Critical point drying of ethanol dehydrated chemically fixed, or freeze-substituted test specimens results in complete loss of discrete polyhead structure. In-lens field-emission scanning electron microscopes and highly sensitive electron detectors are instrumental prerequisites in achieving transmission electron microscope-like resolution of structural details. Shape and size of fine structures, of the test specimen, are accurately imaged at high acceleration voltage ( \u3e 7 kV). Precise localization of antigenic sites via ultra-small (0.8 nm) colloidal gold marker systems by backscattered electrons also depends on the appropriate choice of acceleration voltage. Contamination of the specimen surface is a serious problem in high resolution scanning electron microscopy. It can be controlled in practice, by photographing selected areas of cooled specimens during the first scan of the electron beam

Progress in Scanning Electron Microscopy of Frozen-Hydrated Biological Specimens

Modem scanning electron microscopy yields structural information down to 2 to 5 nm from thin, beam transparent biological specimens. This paper examines the possibilities of garnering this level of structural information from bulk, frozen-hydrated samples. Freeze-fractured, frozen-hydrated yeast cells, frequently taken as a yardstick to monitor progress in low-temperature scanning electron microscopy, have been used to optimize both metal shadowing methods and observation parameters (e.g. accelerating voltage, electron beam irradiation of the specimen). Uncoated frozen-hydrated yeast cells do not charge electrically at an accelerating voltage of 30 kV. Increasing charging effects are however observed with decreasing accelerating voltages. Very thin metal films are therefore used for specimen coating to localize and enhance the specific secondary electron signal. Planar-magnetron sputtering of a 1 nm metal layer provides high resolution secondary electron images, at 30 kV, of freeze-fractured, frozen-hydrated yeast cells in an in-lens field-emission scanning electron microscope. Structural information comparable to that of transmission electron microscopy of freeze-fractures is attained. Planar-magnetron sputtering of either chromium, tungsten or platinum results in essentially the same information density (smallest visible significant structural detail). Frozen-hydrated samples are very beam sensitive and have to be observed under minimum dose conditions