The Role of Ethylene in the Inhibition of Rooting under Low Oxygen Tensions

A 60-fold increase in ethylene content was observed in stem cuttings of chrysanthemum (Chrysanthemum × morifolium Ramat.) held in aero-hydroponics under anoxic conditions during the 8 to 12 days necessary for adventitious root formation. Ethylene, 1-aminocyclopropane-1-carboxylic acid, and 10-(malonylamino) cyclopropane-1-carboxylic acid contents were highest in the immersed portion of the cuttings, but there was substantial ethylene produced by the anoxic, misted portions of the cutting above the liquid. Application of ethylene (10 microliters per liter) to chrysanthemum cuttings stimulated root development in cuttings held in high dissolved oxygen concentrations (8.0 milligrams per liter). Since the application of ethylene did not inhibit rooting in cuttings held at low dissolved oxygen concentrations (2.0 milligrams per liter), the inhibition of rooting under low oxygen concentrations is not mediated by the observed increase in endogenous ethylene content.     

Southern blot analysis of HLA-DP gene polymorphisms in Caucasoid rheumatoid arthritis (RA) patients and controls

Despite extensive analysis of the incidence ofHLA-DR andHLA-DQ allele frequencies in defined autoimmune disease groups, there is very little information available onHLA-DP allele frequencies. This is largely becauseHLA-DP typing has until recently been restricted to primed lymphocyte typing (PLT). However, allelic polymorphism of theHLA-DP subregion can now be studied by Southern blot analysis or genotyping withDPA1 andDPB1 probes. By direct counting of allele-specific DNA fragments, we have analyzed the frequencies of five majorDP genotypes (DPw1, DPw2, DPw3/6, DPw4, andDPw5), in a large number of Caucasoid rheumatoid arthritis (RA) patients (n=74), and controls (n=91). The predicted frequency ofDP alleles in both patient and control groups was comparable to PLT-determinedDP allele frequencies in normal Caucasoids. However, the gene frequency ofDPw4 was increased in the RA patients, with 51% of the patients studied scoring asDPw4, 4 homozygotes. With the exception of one possible combination (DPw5 andDRw6) in the controls, no significant linkage disequilibrium was detected betweenDP andDR alleles in either patient or control groups. Thus the prevalence ofDPw4 in the RA patients is independent of any disease association with theDR loci, and may represent a new class II association with RA.     

Purinergic modulation of cardiac activity in the flounder during hypoxic stress

Summary The interaction of adrenaline and adenosine was examined in cardiac tissue of the flounderPlatichthys flesus.When applied alone both agents increased contractility in both auricular and ventricular myocardial strips. This positive inotropic effect was associated with a small depolarization in the tissues examined by the sucrose gap technique. Simultaneous application of adrenaline and adenosine gave an inhibition of the control responses seen with either agent alone in both auricle and ventricle.Radiocalcium flux studies on ventricular tissue showed that influx was increased by adrenaline or adenosine alone above control values, but when applied together radiocalcium influx was reduced. Radiocalcium efflux from cardiac microsomes was stimulated by challenge with adrenaline or adenosine alone. This stimulation was not seen following simultaneous challenge by both agents.The effect of adrenaline on responses of hypoxic flounder hearts was less than that seen in normoxic hearts. This situation was reversed by pretreatment with the purine receptor blocker caffeine. Caffeine pretreatment also reduced the positive inotropic effect seen in normoxic hearts challenged with adenosine.TLC studies gave strong evidence that hearts perfused with hypoxic salines produced both adenosine and adrenaline.The results are discussed as evidence for a mechanism of heart regulation which the flounder may use as a defence against severe acute hypoxic stress.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: I. Process concept and enzyme binding studies for pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase.     

Early inductive interactions are involved in restricting cell fates of mesomeres in sea urchin embryos

Isolated intact caps of animal blastomeres, obtained from either 8- or 16-cell embryos, differentiate as swollen ectodermal vesicles. These findings agree with earlier studies demonstrating that mesomeres contribute only to larval ectoderm during normal development. In contrast, we find that pairs of mesomeres isolated from 16-cell embryos can differentiate endodermal and mesenchymal cells in a substantial number of cases (23%). Thus, mesomeres have a greater developmental potential than is realized during normal development. Further results support hypotheses that graded distributions of morphogenetic determinants exist within these embryos, since the extent of differentiation of isolated mesomeres is related to the relative position of the third cleavage plane along the animal-vegetal axis. When the third cleavage plane is subequatorial and the resulting animal blastomeres inherit a fraction of the vegetal hemisphere, more cases (39%) differentiate endodermal and mesenchymal cell types. A significant number of mesomere pairs (9-14%), however, can still differentiate endodermal and mesenchymal cells when the mesomeres are formed within the animal hemisphere. Thus, putative vegetal morphogenetic determinants may extend into the animal hemisphere in some cases. Further results indicate a temporal restriction in the developmental potential of mesomeres or mesomere progenitor cells since their differentiative capability is greater if they are isolated earlier during development. Aggregates of isolated mesomere pairs also display a decreased developmental potential when compared to isolated mesomere pairs. These results suggest that associations with adjacent cells (vegetal cells as well as adjacent mesomeres) restrict the development of mesomeres between third and sixth cleavages.     

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.     

Mitogen-responsive S6 kinase

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin- cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K+ concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent Mr of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56,000 (determination in the presence of 200 mM KCl in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase II, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogen-responsive S6 kinase(s) [Martini, O. H. W. and Lawen, A. (1985) in Hormones and cell regulation (Dumont, J. E., Hamprecht, B. and Nunez, J., eds) vol. 9, pp. 411-412, Elsevier Company, North-Holland, Amsterdam; Lawen, A. and Martini, O. H. W. (1985) FEBS Lett. 185, 272-276].     

Insulin-induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase II and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum- and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitro transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphorylate the (principal) S6 site recognized by the cAMP-dependent protein kinase.     

Linkage and the Maintenance of Heritable Variation by Mutation-Selection Balance

The role of linkage in influencing heritable variation maintained through a balance between mutation and stabilizing selection is investigated for two different models. In both cases one trait is considered and the interactions within and between loci are assumed to be additive. Contrary to most earlier investigations of this problem no a priori assumptions on the distribution of genotypic values are imposed. For a deterministic two-locus two-allele model with recombination and mutation, related to the symmetric viability model, a complete nonlinear analysis is performed. It is shown that, depending on the recombination rate, multiple stable equilibria may coexist. The equilibrium genetic and genic variances are calculated. For a polygenic trait in a finite population with a possible continuum of allelic effects a simulation study is performed. In both models the equilibrium genetic and genic variances are roughly equal to the house-of-cards prediction or its finite population counterpart as long as the recombination rate is not extremely low. However, negative linkage disequilibrium builds up. If the loci are very closely linked the equilibrium additive genetic variance is slightly lower than the house-of-cards prediction, but the genic variance is much higher. Depending on whether the parameters are in favor of the house-of-cards or the Gaussian approximation, different behavior of the genetic system occurs with respect to linkage.