Peroxisomal beta-oxidation enzyme proteins in the Zellweger syndrome

The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very long-chain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal beta-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low Mr were seen in the patients. With anti-(oxoacyl-CoA thiolase), high Mr cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal beta-oxidation enzymes.     

Lipid molecular shape affects erythrocyte morphology: a study involving replacement of native phosphatidylcholine with different species followed by treatment of cells with sphingomyelinase C or phospholipase A2

In a previous report it was shown that the replacement of native erythrocyte phosphatidylcholine (PC) with different PC species which have defined acyl chain compositions can lead to morphological changes (Kuypers, F.A., W. Berendsen, B. Roelofsen, J. A. F. Op den Kamp, and L.L.M. van Deenen, 1984, J. Cell Biol., 99:2260-2267). It was proposed that differences in molecular shape between the introduced PC species and normal erythrocyte PC caused the membrane to bend outwards or inwards, depending on the shape of the PC exchanged. To support this proposal, two requirements would have to be fulfilled: the exchange reaction would take place only with the outer lipid monolayer of the erythrocyte, and the extent of lipid transbilayer movement would be restricted. If this theory is correct, any treatment causing unilateral changes in lipid molecular shape should lead to predictable morphological changes. Since this hypothesis is a refinement of the coupled bilayer hypothesis, but so far lacks experimental support, we have sought other means to change lipid molecular shape unilaterally. Shape changes of human erythrocytes were induced by the replacement of native PC by various PC species using a phosphatidylcholine-specific transfer protein: by hydrolysis of phospholipids in intact cells using sphingomyelinase C or phospholipase A2, and by the combination of both procedures. The morphological changes were predictable; additive when both treatments were applied, and explicable on the basis of the geometry of the lipid molecules involved. The results strongly support the notion that lipid molecular shape affects erythrocyte morphology.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Presence of markers for virulence in the unique short region or repeat region or both of pseudorabies hybrid viruses.

The unique short region and part of the repeat region of virulent pseudorabies virus strain NIA-3 was replaced by the corresponding region of the avirulent NIA-4 strain by transfection with subgenomic DNA fragments. The resulting hybrid virus showed a reduced virulence in both mice and pigs. Therefore, important markers for virulence are located in the unique short or repeat region or both of pseudorabies virus. We provide evidence that the terminally located repeat is not required for the generation of progeny with intact pseudorabies virus genomes. Apparently, the terminal repeat is regenerated from the internal repeat.     

Free space iron pools in roots: generation and mobilization

A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaseolus vulgaris L. var. Prélude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum [Thunb.] Jacques). No significant pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel.     

Effect of hypothermia on cell kinetics and response to hyperthermia and X rays

Hyperthermia is a potent radio enhancer. Studies using hypothermia in combination with irradiation have given confusing results due to lack of uniformity in experimental design. This report shows that hypothermia might have potential significance in the treatment of malignant cells with both thermo- and radiotherapy. Reuber H35 hepatoma cells, clone KRC-7 were used to study the effect of hypothermia on cell kinetics and subsequent response to hyperthermia and/or X rays. Cells were incubated at 8.5 degrees C or between 25 and 37 degrees C for 24 hr prior to hyperthermia or irradiation. Hypothermia caused sensitization to both hyperthermia and X rays. Maximum sensitization was observed between 25 and 30 degrees C and no sensitization was found at 8.5 degrees C. At 25 degrees C maximum sensitization was achieved in approximately 24 hr, cell proliferation was almost completely blocked, and cells gradually accumulated in the G2 phase of the cell cycle. In contrast to the effect of hypothermia on either hyperthermia or X rays alone, thermal radiosensitization was decreased in hypothermically pretreated cells (24 hr at 25 degrees C) compared to control cells (37 degrees C). The expression of thermotolerance and the rate of development at 37 degrees C after an initial heating at 42.5 degrees C were not influenced after preincubation at 25 degrees C for 24 hr. The expression of thermotolerance for heat or heat plus X rays during incubation at 41 degrees C occurred in a significantly smaller number of cells after 24 hr preincubation at 25 degrees C. The enhanced thermo- and radiosensitivity in hypothermically treated cells disappeared in approximately 6 hr after return to 37 degrees C.     

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.     

The effect of pH and lysosomal extract on rat liver metallothionein (Cd-thionein)

Was studied in vitro. The¹⁰⁹Cd-labeled protein was isolated by gel filtration and incubated with a lysosomal extract from the same source. No degradation was seen when the pH of the incubation medium was 5 or higher, whereas the degradation of Cd-thionein was completed in 2 h at a pH of 4.5. Dissociation of Cd²⁺ from thionein, which takes place to a fair extent at a pH below 5, appears necessary before the enzymatic breakdown of the thionein moiety can take place.     

Fluorescence decay of pyrene in small and large unilamellar L,α-Dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature

The fluorescence decays of pyrene in small and large unilamellar L,-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10^-7 cm²/s at 40°C to 2.2x10^-6 cm²/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10^-9 cm²/s at 20°C to 7.9x10^-8 cm²/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.Abbreviations SUV small unilamellar vesicles - LUV large unilamellar vesicles - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - FRAP fluorescence recovery after photobleaching Part of this research has been presented at the 5th international symposium on surfactants in solution. Bordeaux, July 9th–13th 1984