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.     

Cellular components of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: immunohistochemical (MHC class II) and electron-microscopic observations

This report deals with the distribution, morphology and specific topical relationships of bone-marrow-derived cells (free cells) in the spinal meninges and dorsal root ganglia of the normal rat. The morphology of these cells has been studied by transmission and scanning electron microscopy. Cells expressing the major histocompatibility complex (MHC) class II gene product have been recognized by immunofluorescence. At the level of the transmission electron microscope, free cells are found in all layers of the meninges. Many of them display characteristic ultrastructural features of macrophages, whereas others show a highly vacuolated cytoplasm and are endowed with many processes. These elements lack a conspicuous lysosomal system and might represent dendritic cells. Scanning electron microscopy has revealed that free cells contact the cerebrospinal fluid via abundant cytoplasmic processes that cross the cell layers of the pia mater and of the arachnoid. Cells expressing the MHC class II antigen are also found in all layers of the meninges. They are particularly abundant in the layers immediately adjacent to the subarachnoid space, in the neighbourhood of dural vessels, along the spinal roots and in the dural funnels. In addition to the meninges, strong immunoreactivity for MHC class II antigen is observed in the dorsal root ganglia. The ultrastructural and immunohistochemical findings of this study suggest the existence of a well-developed system of immunological surveillance of the subarachnoid space and of the dorsal root ganglia.     

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3 end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GC-I8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC isoelectric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.