Small-angle X-ray study of DNA-dependent RNA polymerase subunit sigma from Escherichia coli.
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Biomedical subjects
Publications and source records attributed to H Heumann.
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This study investigated the effects of enbucrilate (Histoacryl) on the middle ear. Small amounts of the adhesive were used in 81 middle-ear operations performed on 50 rabbits. Light microscopic investigation of these ears 1, 2, and 3 months after surgery showed bone necrosis and a granulation tissue reaction surrounded by foreign-body giant cells. Inner-ear changes were observed in a third of the cases. Therefore, enbucrilate should only be used with extreme care and in minute quantities and never near the labyrinthine capsule.
The core subunit arrangement of alpha 2-beta-beta' within DNA-dependent RNA polymerase holoenzyme alpha 2 beta beta' sigma from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits, alpha 2, beta and beta' in situ and for the determination of the intersubunit centre-to-centre distances. The contrast between the buffer and the remaining 'hydrogenated' enzyme vanishes if the buffer contains 42% 2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved. The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering intensity differences of the pair-labelled (e.g. both beta and beta' labelled) and both mono-labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains sigma as a dissociable factor, is presented in a three-dimensional model.
The complete 'centre-of-subunit structure' of the multisubunit enzyme DNA-dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre-to-centre distances d between the core subunits alpha 2, beta and beta' presented in the preceding paper, the values of d between initiation factor sigma and alpha 2 (8.4 +/- 1.6 nm), beta (4.4 +/- 2.2 nm) and beta' (10.7 +/- 1.5 nm) were derived from R of sigma (4.1 +/- 0.3 nm) in situ and of the pairs alpha 2--sigma (6.1 +/- 0.4 nm), beta--sigma (5.6 +/- 0.3 nm) and beta'--sigma (7.5 +/- 0.4 nm) within the holoenzyme (alpha 2 beta beta' sigma). The structural parameters of the subunits within their molecular complex are accessible for neutron small-angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of 'hydrogenated' molecular parts and extended exposure times because of weak scattering effects. The overall shape of sigma bound to core enzyme (alpha 2 beta beta') proved to be identical (within experimental resolution) with sigma in the isolated state measured recently by X-ray small-angle scattering. The refined shape of isolated sigma was reduced to an ellipsoid which was orientated with respect to the core structure (alpha 2--beta--beta') in a 'space-filling' way around the position of the sigma centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three-dimensional model.
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Platelets of healthy test persons were gained according to the method of Aster (avital cells) and according to the procedure of BUBE and GMURZYNSKI (vital cells). Because of their tendency towards aggregation vital cells do bind any HL-A antibodies and whereas avital cells will fix antibodies and complement depending on the height of the titre. Thrombocytes stored in the own serum will respond less to the micro-C'-fixation test at + 4 degrees C as well as--196 degrees C, whereas storage in NaN3/NaCl and under the conditions of the own serum will reveal no differences up to--196 degrees C in each case.
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The influence of papain, neuraminidase and AET to lymphocytes is reported. All three substances strengthen the lymphocytotoxic reaction in low concentrations or by short incubation time and lower it in higher concentrations or longer incubation since. Membrane changes by these three substances are reversible under defined conditions.
Experiments were performed on 19 mongrel dogs. Changes of transplantation antigens by anesthesia and by brain ischemia were identified in cytotoxic tests. Brain ischemia was responsible for the significant alterations in 5 antigens, anesthesia alone induced significant changes in 3 antigens.
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