Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-α has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling. © 1994 Wiley-Liss, Inc.     

Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer-dimer interaction.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.     

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

Expression of an Arabidopsis potassium channel gene in guard cells.

The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.     

Ecological distinctions between sympatric species of Saguinus and Sciurus

Tamarins are small New World monkeys that have been described as "squirrellike." Squirrels, along with bats and birds, are the taxa most likely to utilize resources similar to those used by primates in the tropical forest canopy. In this paper we compare differences in ecology, diet, locomotion, and habitat utilization between sympatric populations of tamarins (Saguinus oedipus) and tree squirrels (Sciurus granatensis) in Panama. Data presented indicate that although there is some degree of resource overlap, patterns of habitat utilization differ significantly. Rather than being "squirrellike," the Panamanian tamarin exhibits a pattern of locomotor and feeding behavior consistent with that found in other arboreal primates.     

Modulation of the cAMP relay in Dictyostelium discoideum by ammonia and other metabolites: possible morphogenetic consequences

Using a perfusion technique (P.N. Devreotes, P.L. Derstine, and T.L. Steck, 1979, J. Cell Biol. 80, 291-299), it has been shown that cAMP secretion by aggregation-competent cells in response to an exogenous cAMP signal is significantly reduced by exposure to NH4Cl or any of a set of carboxylic acids that includes propionate, succinate, pyruvate, and acetate. The effects of NH4Cl and any of the carboxylic acids are additive and the combinations restrict cAMP secretion to barely detectable or insignificant levels. The inhibitions are rapidly expressed, and are reversible. The activity of NH4Cl is marked at pH 7.2 and undetectable at pH 6.2. Hence, NH3 is presumably the active molecular species. Propionate activity is significantly greater at pH 6.2 than 7.2, indicating that the un-ionized acid is the active species. The data presented herein indicate that these effects are exerted via two separate and independent routes. During exposure of cAMP-stimulated cells to NH4Cl, the decrease in intracellular cAMP accumulation was even greater than the decrease in extracellular accumulation. Hence, NH3 appears to act as a cAMP accumulation inhibitor (CAI). In contrast, exposure to carboxylic acid concentrations that drastically reduce extracellular cAMP accumulation can actually enhance or, at worst, only slightly reduce intracellular accumulation. Hence, the carboxylic acids appear to act as cAMP release inhibitors (CRI). Stationary phase cells incubated on solid substratum in the presence of NH4Cl plus succinate (or propionate) for 18 hr failed to exhibit even the earliest signs of aggregation. If then harvested and redeposited in the absence of the metabolites, they proceeded through the morphogenetic sequence with approximately normal kinetics, suggesting that no significant morphogenetic competence had been achieved during their previous tenure. The morphogenetic implications of cAMP relay modulation are discussed.     

Reversible inhibition of aggregation-related cohesivity in Dictyostelium discoideum by diffusible metabolites

Evidence presented elsewhere (G.B. Williams, E.M. Elder, and M. Sussman 1984, Dev. Biol. 105, 377-388) indicates that NH3 and certain carboxylic acids including propionate, succinate, and acetate modulate the cAMP relay in Dictyostelium discoideum. The former appears to act as a cAMP accumulation inhibitor, the latter as cAMP release inhibitors. The cohesive properties of aggregation competent cells have been assayed quantitatively in the presence of these modulators. The following results were obtained: (1) At pH 7.5, EDTA-resistant cohesivity was greatly inhibited by NH4C within the concentration range tested (30-3.8 mM). Even at the higher concentrations the effect was not immediate but required ca. 10 min for full expression. At the lower concentrations, the inhibitory level was only slightly reduced but the time for full expression progressively increased. At pH 6.5, the level of inhibition was marginal, indicating that NH3 is the active molecular species. By themselves, neither ambient pH nor ionic strength appeared to affect cohesive performance within the ranges employed. The inhibition was immediately and completely reversed upon removal of NH4Cl or a shift of ambient pH from 7.5 to 6.5. The presence of cycloheximide did not affect the recovery of cohesivity after NH4Cl removal. (2) The presence of 15 mM succinate, propionate, or acetate also reduced cell cohesivity. The timing and extent of the inhibition were identical at pH 7.5 and 6.5. The inhibition was expressed immediately and was reversible. Each of the acids acted synergistically with NH4Cl. The relative potencies of these metabolites acting singly or in combination as inhibitors of cohesivity corresponded roughly to their potencies as modulators of the cAMP relay (Williams et al., 1984). (3) The sensitivity to the metabolites was stage specific, being maximal during and shortly after aggregation and disappearing abruptly at 11-12 hr. This corresponds to the time at which this cohesive system, responsible for the end-to-end cell associations evident during aggregation (H. Beug, G. Gerisch, S. Kempff, V. Riedel, and G. Cremer, 1970, Exp. Cell. Res. 63, 147-158) is supplanted by a newly arisen, serologically and genetically distinct system which thereafter maintains the integrity of the aggregate (C. Steinemann and R.W. Parish, 1980, Nature (London) 286, 721-724; D.K. Wilcox and M. Sussman, 1981, Dev. Biol. 82, 102-112, and Proc. Natl. Acad. Sci. USA 78, 358-362; C.L. Saxe III and M. Sussman, 1982, Cell 29, 755-759). The activities of the metabolites, detailed above, are discussed in relation to their previously demonstrated activities as morphogens.     

Induction of stage-specific cell cohesion in D. discoideum by a plasma-membrane-associated moiety reactive with wheat germ agglutinin

Dictyostelium discoideum strain JC-5 is a temperature-sensitive, cohesion-defective mutant. At the restrictive temperature, aggregates are formed and develop normally to a specific late stage, but then disperse to a lawn of noncohesive cells. The defect stems from the inability of the mutant at the restrictive temperature to accumulate or retain an adequate level of a newly arisen, plasma-membrane-associated cohesive moiety during the later stages of morphogenesis. We now report that the noncohesive mutant cells can be induced to cohere at a level of greater than 99% by addition of a fraction reactive with wheat germ agglutinin and isolated from the plasma membranes of late-stage, cohesive mutant or wild-type cells. Furthermore, the cohesivities of late-stage, cohesive mutant or wild-type cells can be significantly enhanced by the same preparations but early-stage cohesive cells remain unaffected. The cohesion-inducing activity can be recovered from two regions of a one-dimensional polyacrylamide gel electropherogram following elution and renaturation. The first is associated with a single silver-staining band (apparent molecular weight 95,000). It appears de novo at a postaggregative stage, and accumulates in a thermostable fashion in the wild-type and in a thermosensitive fashion in the mutants, concurrent with the loss of cohesivity. The second is associated with a complex of silver-staining bands (apparent molecular weights 40,000-50,000). The activity of the eluate reactive with wheat germ agglutinate is destroyed within 5 min at 100 degrees C.