Telomere sequences attached to nuclearly migrated yeast linear plasmid

The yeast linear plasmid pCLU1, derived from pGKL1, has terminal proteins (TPs) covalently attached at the 5' ends of inverted terminal repeats (ITRs) and replicates in the cytoplasm, presumably using the TP as a primer for DNA synthesis. In Saccharomyces cerevisiae, under certain conditions, pCLU1 migrated into the nucleus and replicated in either linear or circular form. The linear-form plasmid lacked TPs; instead it carried host-telomere repeats at the ITR ends. The present study showed that (1) the added telomere was primarily composed of the repeated tracts of TGTGTGGGTGTGG, which was complementary to the RNA template of yeast telomerase, (2) the telomeric addition occurred at the very end of the ITRs, and (3) the sequence composition of the added telomeres was diverse among individual plasmids, but symmetrically identical at both ends of each plasmid. A similar mode of telomere addition was also observed in cells defective in the RAD52 gene.     

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.     

DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA^Gln and tRNA^Glu respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S‐phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate‐limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.     

Studies on the NADH-menaquinone oxidoreductase segment of the respiratory chain in Thermus thermophilus HB-8

Five distinct low potential iron-sulfur clusters have been identified potentiometrically in the membrane particles from Thermus thermophilus HB-8. Three of these clusters (designated as [N-1H]T, [N-2H]T, and [N-3]T) exhibit the following midpoint redox potentials and g values (Em8.0 = -274 mV, gx,y,z = 1.93, 1.94, 2.02), (Em8.0 = -304 mV, gx,y,z = 1.89, 1.95, 2.04), and (Em8.0 = -289 mV, gx,y,z = 1.80, 1.83, 2.06), respectively. These clusters, one binuclear and two tetranuclear, have been shown to be components of the energy coupled NADH-menaquinone oxidoreductase complex (NADH dh I). They are reducible by NADH in the piericidin A-inhibited aerobic membrane particles as well as in the purified NADH dh I complex. Two additional very low potential iron-sulfur clusters (one binuclear, [N-1L]T, and one tetranuclear, [N-2L]T) were observed in membrane particles. These clusters possess the following physiochemical properties (Em8.0 = -418 mV, gx,y,z = 1.93, 19.5, 2.02) and (Em8.0 = -437 mV, gx,y,z = 1.89, 1.95, 2.04), respectively. No high potential tetranuclear cluster equivalent to the mitochondrial iron-sulfur cluster [N-2]B was found in this bacterial system. In membrane particles isolated from T. thermophilus HB-8 cells, four different semiquinone species have been identified based on their redox midpoint potentials [Em9(Q/QH2) = 40, -100, -160, -300 mV] and sensitivity to the quinone analogue inhibitor, 2-heptyl-4-hydroxy quinoline-N-oxide. Of these semiquinone species the -100 mV component has been suggested to be part of the NADH dehydrogenase. Piericidin A sensitive delta psi formation has been demonstrated to be coupled to the NADH-MQ1 oxidoreductase in membrane vesicles of T. thermophilus HB-8.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Induction of DNA damage and apoptosis in Saccharomyces cerevisiae by a yeast killer toxin

The cellular response of Saccharomyces cerevisiae to a linear plasmid encoded killer toxin from Pichia acaciae was analysed. As for the Kluyveromyces lactis zymocin, such toxin was recently shown to bind to the target cell's chitin and probably acts by facilitating the import of a toxin subunit. However, as distinct from zymocin, which arrests cells in G1, it provokes S-phase arrest and concomitant DNA damage checkpoint activation. Here, we report that such novel toxin type causes cell death in a two-step process. Within 4 h in toxin, viability of cells is immediately reduced to approximately 30%. Elevated mutation rates at the CAN1 locus prove DNA damaging mediated by the toxin. Cells arrested artificially in G1 or G2/M are very rapidly affected, while cells arrested in S loose their viability at a slower rate. S-phase arrest is, thus, a response of target cells to cope with DNA damage induced by the toxin. A second decline in viability requiring metabolically active target cells emerges upon toxin exposure over 10 h. During this phase, toxin treated cells develop abnormal nuclear morphology and react positive to terminal deoxynucleotidyl transferase-mediated nick end-labelling (TUNEL), indicative of DNA fragmentation. Furthermore, as judged from staining with fluorescein conjugated annexinV, cells expose phosphatidylserine at the outer membrane face and the formation of reactive oxygen species (ROS) is increased. ROS formation and concomitant cell death was heavily suppressed in a rho- derivative of the tester strain, while immediate reduction of viability was indistinguishable from the wild type. As a strain lacking the cellular target because of defects in the major chitinsynthase (Chs3) did not display such characteristic changes, the chitin binding and DNA-damaging P. acaciae toxin constitutes an apoptosis inducing protein. Both, DNA-damaging and apoptosis induction are unique features of this novel toxin type.