Changes in cellular polyamine contents and activities of their biosynthetic enzymes at each phase of the cell cycle in by-2 cells

We analyzed changes in polyamine contents and the activities of biosynthetic enzymes during each phase of the cell cycle for a synchronized population of BY-2 cells. Based on our analysis of H³-thymidine incorporation flow cytometry, and the mitotic index, the M and G₂ phases seemed to occur at 8 h and from 2.5 to 8 h, respectively, after the release of aphidicolin. The respective activities of arginine decarboxylase (ADC), Ornithine decarboxylase (ODC), and S-adenosyl methionine decarboxylase (SAMDC) at the beginning (7.4, 11.2, and 5.5 nmol mg^-1 protein h^-1) were increased to 22.6, 22.1, and 15.1 nmol mg^-1 protein h^-1. However, those increases do not coincide with the general change in polyamines reported from animal cells. In addition, the bi-phasic activation of polyamine biosynthetic enzymes, such as those found in the general animal model, was observed with ADC and ODC but not with SAMDC. These results suggest that the general animal model for explaining polyamine changes and SAMDC activation in the cell cycle cannot be applied to BY-2 cells. Further, our flow-cytometric analysis of cell populations may be a useful tool for evaluating the effects of polyamines on cell cycle progression in BY-2 cells.     

Parsing the effects of binding,signaling, and trafficking on the mitogenic potencies of granulocyte colony-stimulating factor analogues

The pharmacodynamic potency of a therapeutic cytokine interacting with a cell-surface receptor can be attributed primarily to three central properties: [1] cytokine/receptor binding affinity, [2] cytokine/receptor endocytic trafficking dynamics, and [3] cytokine/receptor signaling. Thus, engineering novel or second-generation cytokines requires an understanding of the contribution of each of these to the overall cell response. We describe here an efficient method toward this goal in demonstrated application to the clinically important cytokine granulocyte colony-stimulating factor (GCSF) with a chemical analogue and a number of genetic mutants. Using a combination of simple receptor-binding and dose-response proliferation assays we construct an appropriately scaled plot of relative mitogenic potency versus ligand concentration normalized by binding affinity. Analysis of binding and proliferation data in this manner conveniently indicates which of the cytokine properties-binding, trafficking, and/or signaling-are contributing substantially to altered potency effects. For the GCSF analogues studied here, two point mutations as well as a poly(ethylene glycol) chemical conjugate were found to have increased potencies despite comparable or slightly lower affinities, and trafficking was predicted to be the responsible mechanism. A third point mutant exhibiting comparable binding affinity but reduced potency was predicted to have largely unchanged trafficking properties. Surprisingly, another mutant possessing an order-of-magnitude weaker binding affinity displayed enhanced potency, and increased ligand half-life was predicted to be responsible for this net beneficial effect. Each of these predictions was successfully demonstrated by subsequent measurements of depletion of these five analogues from cell culture medium. Thus, for the GCSF system we find that ligand trafficking dynamics can play a major role in regulating mitogenic potency. Our results demonstrate that cytokine analogues can exhibit pharmacodynamic behaviors across a diverse spectrum of "binding-potency space" and that our analysis through normalization can efficiently elucidate hypotheses for the underlying mechanisms for further dedicated testing. We have also extended the Black-Leff model of pharmacological agonism to include trafficking effects along with binding and signaling, and this model provides a framework for parsing the effects of these factors on pharmacodynamic potency.     

Relations between and inheritance of chlorogenic acid contents in an interspecific cross between Coffea pseudozanguebariae and Coffea liberica var ‘dewevrei’

 Chlorogenic acids (CGA) are phenolic compounds commonly found in green coffee beans. The main CGA classes are caffeoylquinic acids (CQA), dicaffeoylquinic acids (diCQA), and feruloylquinic acids (FQA). Each contains three isomers differing in the number and identity of the acylating residues. An interspecific cross between Coffea pseudozanguebariae (low CGA content) and C. liberica var ‘dewevrei’ (high CGA content) was investigated for CGA contents in F₁ and back-cross hybrids. Relations within and between CGA classes were studied and confirmed the known biosynthesis pathway. A single major gene was noted for the 3-FQA isomer; absence was dominant. Additivity was found for most other isomers either with or without the transformation of variables. Conversely, most ratios were not additive, due to a curvilinear relation between some isomers. The consequences for breeding both in terms of cup taste improvement and disease resistance are discussed. Received: 20 July 1998 / Accepted: 14 August 1998     

Imprinting capacity of gamete lineages in Caenorhabditis elegans

We have observed a gamete-of-origin imprinting effect in C. elegans using a set of GFP reporter transgenes. From a single progenitor line carrying an extrachromosomal unc-54::gfp transgene array, we generated three independent autosomal integrations of the unc-54::gfp transgene. The progenitor line, two of its three integrated derivatives, and a nonrelated unc-119:gfp transgene exhibit an imprinting effect: single-generation transmission of these transgenes through the male germline results in approximately 1.5- to 2.0-fold greater expression than transmission through the female germline. There is a detectable resetting of the imprint after passage through the opposite germline for a single generation, indicating that the imprinted status of the transgenes is reversible. In cases where the transgene is maintained in either the oocyte lineage or sperm lineage for multiple, consecutive generations, a full reset requires passage through the opposite germline for several generations. Taken together, our results indicate that C. elegans has the ability to imprint chromosomes and that differences in the cell and/or molecular biology of oogenesis and spermatogenesis are manifest in an imprint that can persist in both somatic and germline gene expression for multiple generations.     

Folding of A+U-rich RNA elements modulates AUF1 binding. Potential roles in regulation of mRNA turnover

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg(2+) induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg(2+)-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg(2+) and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.     

Dusts causing pneumoconiosis generate .OH and produce hemolysis by acting as Fenton catalysts

Silicates causing pneumoconiosis function as Fenton catalysts to generate hydroxyl radicals (.OH) when incubated with hydrogen peroxide and a reducing substance. In contrast, silicates which do not cause pneumoconiosis demonstrate no Fenton activity. Catalytic activity is decreased by pretreatment of silicates with the iron chelators deferoxamine or transferrin. Hemolysis from silicates is decreased by interventions which remove superoxide anion or hydrogen peroxide from the medium, or by pretreatment of dusts with iron chelators. Thus, asbestos and nonfibrous silicates may cause pneumoconiosis through a common oxidant mechanism by catalyzing production of toxic .OH radicals in the lung.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.