Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

The effect of (+) cyanidanol-3 on the Na+-K+-ATP-ase and Mg++-ATP-ase activities of the rat brain in the presence and absence of ascorbic acid

The authors investigated the effects of (+) cyanidanol-3-(Catergen) in vitro on the activities of rat brain plasma membrane and microsomal Na+-K+-ATP-ase and Mg++-ATP-ase, in the presence and absence of ascorbic acid. Due to lipid peroxidation induced by low concentration of ascorbid acid, activity of both ATP-ase decreased. (+) cyanidanol-3 proved to be an effective antioxidant in this system. It inhibited the decrease of ATP-ase activity which occurred as a result of lipid peroxidation promoted by ascorbic acid.     

The effect of pentylenetetrazol on the metacerebral neuron of Helix pomatia

The effects of Pentylenetetrazol (PTZ) on the metacerebral giant cell (MCC) of the snail, Helix pomatia were studied. Actions on membrane resistance, time constant, resting and action potentials, outward and inward ionic currents were examined. Superfusion with PTZ in concentrations of 25 to 50 mmol/l, induced a gradually evolving convulsive state, which could be studied by intracellular recording from the MCCs. In the pre-convulsive state an acceleration of the spontaneous activity developed and was followed by paroxysmal depolarization shifts (PDSs), in the convulsive phase. PTZ prolonged the membrane time constant by about 10 percent, but this could not be traced back to alterations in membrane resistance or capacity. The resting membrane potential was not significantly altered; the action potentials were prolonged by slowing down of both the rising and decaying phases. The outward potassium currents were repressed by PTZ in a voltage dependent manner. The decrease of the IA current became more pronounced at increasingly positive command pulses, while IK was relieved from depression especially at longer pulse durations. Inward currents were isolated with the aid of suppression of outward currents by 50 mmol/l TEA. Under these conditions sodium currents, measured in calcium deficient Ringer solution were moderately depressed, while the calcium currents, examined during sodium-free superfusion, were mildly enhanced by PTZ. It is concluded that PTZ effects on ionic conductances, on membrane parameters, on the resting potential and ionic currents explain only modifications of spike potentials occurring in the convulsive state and do not account for the PDS, the central phenomenon of the convulsive electrographic activity, at least in this thoroughly examined type of neuron.     

Standardization of salt aggregation test for reproducible determination of cell-surface hydrophobicity with special reference to Staphylococcus species

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 times 10⁹ cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A × 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20C reduced cell-surface hydrophobicity of all species, while storage at 4C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface.     

Transposon mutagenesis of Rhizobium japonicum

Summary We report here successful mutagenesis with Transposon Tn5 of three slow-growing strains of Rhizobium japonicum USDA 122, 61A76, USDA 74 and one fast-growing strain, USDA 191. Strains were chosen as representatives of different DNA homology and serogroups of this divergent species, which effectively nodulate North American soybean cultivars. The source of Tn5 was the suicide plasmid pGS9, which possesses broad host range N-type transfer genes in a narrow host range p15A replicon. The selection of Tn5 mutants was facilitated by the expression of the Tn5 encoded streptomycin gene in R. japonicum. Kanamycin and streptomycin resistant colonies appeared from interspecific crosses with E. coli at optimal frequencies of 10^-6 for R. japonicum USDA 61A76 and USDA 191 and 5x10^-7 for R. japonicum USDA 122 and USDA 74. Altogether, 6550 Tn5 mutants were isolated in USDA 122 and 61A76, and a small number from USDA 74 and USDA 191. Colony hybridization showed that all tested mutants of 61A76 and USDA 122 contained Tn5. Physical analysis of total DNAs from representative numbers of USDA 122, 61A76 and USDA 191 mutants revealed that each of them carried one copy of the transposon integrated randomly in the genome. This was also true for most USDA 74 mutants. Screening of mutants for auxotrophy showed frequencies of 0.2% for USDA 122 and 0.08% for 61A76. Several symbiotically defective mutants were identified on plants, Glycine soja and G. max.     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.