A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Reversible cross-linking of crystalline bacterial surface layer glycoproteins through their glycan chains

After periodate oxidation and incubation with dithiodipropionic acid dihydrazide cross-linking of the crystalline surface layer (S-layer) glycoproteins of Clostridium thermohydrosulfuricum L111-69 and Bacillus alvei CCM 2051 was achieved specifically through the glycan chains. The cross-linked S-layers were used for the immobilization of chemically synthesized, spacer-linked, tumour-associated T-disaccharide [Gal(13)GalNAc]. Electron microscopical evaluation of the resulting conjugates showed densely packed, multilayered S-layer structures loaded with the immobilized ligand. After reductive cleavage of the disulphide bond of dithiodipropionic acid by dithiothreitol, monomeric haptenated S-layer conjugates could be obtained. Both the cross-linked and the monomeric type of conjugate might be useful for assessment of specific immune responses, which, in general, can be elicited by those artificial antigens. Correspondence to: P. Messner     

Screening for microorganism with cell wall lytic activity to produce protoplast-forming enzymes

Several different bacteria and fungi capable of degrading yeast cell walls were isolated in the course of a screening programme. One Streptomyces and one Acremonium strain were found to degrade yeast cell walls extremely well. Both isolates produced enzymes in liquid culture that could be used for protoplasting of Sporobolomyces salmonicolor (DSM 70851) and Rhodotorula rubra (DSM 70403). This fact is quite remarkable as, so far, S. salmonicolor could not be protoplasted by commercially available enzymes. Correspondence to: W. Kaul     

Different genomic structure of mouse and human Lmp7 genes: characterization of MHC-encoded proteasome genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L11045 (human LMP7) and L11145 (mouse Lmp-7).     

Subtypes of non-transformed human mammary epithelial cells cultured in vitro: histo-blood group antigen H type 2 defines basal cell-derived cells

Abstract. Normal (non-transformed) human mammary epithelial cell lines derived from reduction mammoplasties were analyzed by immunocytochemistry with more than 80 monoclonal antibodies (mAbs) and other specific reagents to tissue-specific and developmentally regulated antigens at different passage levels. A subpopulation of poorly differentiated, proliferating epithelial cells, corresponding to the 'selected' cell type of late passages, is shown to be characterized by a new marker, the histo-blood group antigen H type 2, probably carried on a membrane-bound glycolipid. These cells also express a number of other onco-developmental carbohydrate antigens [Le^y, Le^x, sialosyl-Le^a, precursor of Thomsen Friedenreich antigen (T_n), but not Thomsen-Friedenreich antigen and sialosyl-T_n]. Their cytokeratin (CK) phenotype, as assessed by reactivity with monospecific mAbs and two-dimensional gel electrophoresis, is CK 5, 6, 14 and 17, with CK 19 being consistently absent, and varying minor amounts of CK 7, 8 and 18, as well as 15 and 16. The reactivity of these cells with a panel of 11 mAbs specific for CK 18 varies considerably even after cloning, indicating heterogeneity of epitope expression or accessibility. Our data strongly suggest that the H type 2⁺ cells develop from the basal cell layer of the mammary gland.     

Abundance,horizontal and vertical distribution of fish in eastern Weddell Sea micronekton

Summary The spatial distribution and species composition of high-Antarctic ichthyonekton was investigated during the EPOS 3 cruise by RV Polarstern in the eastern Weddell Sea during January–February 1989. A multiple rectangular midwater trawl was used to collect samples from the surface to near the sea floor at 11 stations along a 245 kra transect off Halley Bay. Early larval stages of 18 species, representing about 24% of the known Weddell Sea ichthyofauna, were present in the water column. The Antarctic silver-fish, Pleuragramma antarcticum, over-whelmingly dominated the catches comprising 84.5% of the 5022 specimens caught. The abundance of this species markedly increased towards the offshore end of the transect with the highest numbers occurring near the shelf-break front associated with the westerly current of the southern limb of the Weddell Gyre. The increased abundance of P. antarcticum in continental slope waters was attributed to deflection of the East Weddell Coastal Current beyond the shelf/slope break by fringing ice shelves. Most larval and juvenile fish were found in the seasonally warmed upper 0–70 m layer of the Antarctic Surface Water where conditions occurred that appeared to be favourable to both feeding and growth. Cluster analysis indicated that inner-, central-and outer-shelf assemblages were represented and that the species composition was most effectively described by reference to water mass and depth.     

Crystalline bacterial cell surface layers

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.