Partial Purification and Immunocytocharacterization of the Plasma Membrane ATPase of Jerusalem artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Plasmalemma ATPase from Jerusalem artichoke tubers was studiedin relation to the dormancy of tubers. After partial purification,one peptide of 110 kDa appeared on SDS PAGE electrophoresisfrom dormant and non-dormant materials. ATPase specific activitywas twice higher on dormant material in the crude and solubilizedfractions, but was the same in both materials after partialpurification. Immunolabeling of this enzyme was made using aspecific antibody raised against the C terminal portion of theH⁺-ATPase from Arabidopsis thaliana. Immunolabeling was morepronounced in dormant material, in vitro and in situ. Severalworks had shown that the C terminal part of the enzyme couldbe involved in its regulation. The results presented are discussedin relation to the hypothesis according to which an internaleffector could modulated the plasmalemma ATPase activity, duringdormancy breaking. (Received October 25, 1993; Accepted September 6, 1994)     

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

Effect of taurine,l-glutamine and l-histidine addition in an amino acid glucose solution on the cellular bioavailability of zinc

Radioactive zinc was used to study the effect of a binary parenteral nutrient solution, composed of amino acids and glucose, on zinc uptake by fibroblasts. The influence of addition of taurine, l-glutamine and of the increase in l-histidine content of the admixture was assessed. The pure mixture was highly toxic for cells and so it was diluted 1/5 in tyrode buffer with 2% albumin. As compared with cells incubated in the buffer containing albumin, zinc absorption was significantly higher (P < 0.05) in the presence of the amino acids of the mixture. Amino acids thus increased bioavailability by displacing zinc bound to albumin. When the histidine concentration in the nutrient medium (4.2 mm) was doubled, inhibition was noted after 30 min of incubation and zinc uptake thereafter remained comparable to that in histidine-free medium. The addition of glutamine (4.2 mm), usually not present in binary mixtures, resulted in significant differences as compared with glutamine-free control medium. Taurine (0.8 mm), led to a constant increase in zinc uptake by fibroblasts as compared with that obtained with taurine-free mixture. However, ultrafiltration showed that taurine was not able to displace zinc from albumin.     

A polymorphic alpha satellite sequence specific for human chromosome 13 detected by oligonucleotide primed in situ labelling (PRINS)

The centromeric alpha satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence and cannot be easily distinguished by mean of probes for Southern blot or in situ hybridisation. We have used the oligonucleotide-primed in situ (PRINS) labelling technique with primers defined from the alpha satellite sequence of chromosome 13. One primer was found to label specifically the centromeric region of chromosomes 13 and allowed the detection of a polymorphism between two chromosome 13 homologues in one individual.     

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.     

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Solubilization and reconstitution of the mitochondrial peptide-sensitive channel

In addition to the voltage-dependent anion channel (VDAC), mitochondrial outer membranes contain a cationic channel of large conductance, which is blocked by a mitochondrial addressing peptide (peptide-sensitive channel, PSC). Bovine adrenal cortex mitochondria were solubilized in 1.5% octyl -glucoside, and membrane vesicles were reconstituted by slow dilution with a low ionic strength buffer. The reconstituted vesicles contained a functional channel possessing the electrical characteristics of the cationic channel, including its sensitivity to the mitochondrial addressing peptide. Important features of the described protocol are the nature of the detergent, its concentration, and the addition of glycerol during the whole procedure. No solubilization could be observed in the presence of cholate.     

Reversal of selenium and zinc deficiencies in chronic hemodialysis patients by intravenous sodium selenite and zinc gluconate supplementation

In six chronic dialyzed uremic patients, an intravenous sodium selenite (Se 50 μg during 5 wk and then 100 μg) and zinc gluconate (Zn 5 mg) supplementation was performed during 20 wk at each dialysis session three times weekly. Before supplementation, plasma Se and Zn, plasma and erythrocytes (RBC) antioxidant metalloenzymes glutathione peroxidase (GPX), and superoxide dismutase (SOD) were significantly decreased, whereas lipid peroxidation (as thiobarbituric acid reactants TBARs) was increased. To obtain a significative change in plasma selenium, we had to use an Se dose of 100 μg/dialysis session. Then, treatment-increased plasma Se (from 0.58 ±0.09 to 0.89±0.16 μmol/L) led to a repletion of RBC-GPX (from 29.6±6 to 43±5.8 U/g Hb) and increased plasma GPX levels (from 62±13 to 151±43 U/L). Plasma Zn and RBC-SOD did not vary significantly. The change of TBARs was not observed between wk 1 and 4. They decreased significantly between wk 4 (4.80±0.21μmol/L) and wk 20 (4.16±0.26 μmol/L). We noted a low correlation between TBARs and plasma GPX. A strong correlation was observed between Se and plasma GPX. The reversal of Se deficiencies should reduce oxidative damage observed in these patients.