A Pro/Ser substitution in nucleoside diphosphate kinase of Drosophila melanogaster (mutation killer of prune) affects stability but not catalytic efficiency of the enzyme.

Nucleoside diphosphate kinase of Drosophila, recently identified as the product of the awd gene, is essential for larval development. The conditional lethal mutation Killer of prune maps to the same gene. We purified the nucleoside diphosphate kinases from wild-type and mutant larvae by a simple procedure involving affinity chromatography on blue Sepharose. Both proteins are purified as hexamers in their native state. The mutant protein, which carries a serine instead of proline at position 97, has structural properties and catalytic efficiency that are very similar to the wild-type protein. However, the mutant protein has a much lower stability to denaturation by heat and urea. Following dilution of urea with buffer the urea-denaturated mutant nucleoside diphosphate kinase accumulates as folded monomers and cannot recover its quaternary structure and enzymatic activity. In contrast, the wild-type enzyme recovers hexameric structure and activity. This suggests that the mutation affects the folding/assembly pathway without affecting the function of the mature protein once folded and assembled into the mature hexameric structure.     

Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition.

In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor.     

Calcium regulation of growth and differentiation in Streptococcus pneumoniae.

Streptococcus pneumoniae requires 0.15 mM-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mM triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2',4'-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.     

Cytokine production after intravenous or peritoneal gram-negative bacterial challenge in mice. Comparative protective efficacy of antibodies to tumor necrosis factor-alpha and to lipopolysaccharide.

The production of TNF-alpha, IL-1, and IL-6 was measured in mice after bolus i.v. Escherichia coli O111 LPS injections and during bacteremia induced either by bolus i.v. or by i.p. challenges of live E. coli O111. High but transient TNF-alpha peaks were observed after bolus i.v. LPS or bacterial challenges. In contrast, the levels during lethal peritonitis increased progressively to values 50- to 100-fold lower than the peak values observed after i.v. injections, and remained sustained until death. Whereas after i.v. challenge with 1000 LD50 of LPS, anti-TNF-alpha antibody fully protected mice from death and reduced serum IL-1 and IL-6 levels, anti-TNF-alpha antibody did not improve the survival of mice nor reduced serum IL-1 and IL-6 levels after i.p. bacterial challenge. In contrast to anti-TNF-alpha antibodies, anti-LPS antibodies were protective in the peritonitis model. Protection was accompanied by a striking reduction of bacterial numbers and of TNF-alpha, IL-1, and IL-6 levels in the serum, but the levels of these cytokines were only marginally affected in the peritoneal lavage fluid. This latter observation demonstrates that the local peritoneal cytokines did not diffuse readily into the circulation, thus suggesting that at least part of the circulating cytokines are produced systemically. In conclusion, the striking differences between cytokine profiles as well as the divergent efficacy of anti-TNF-alpha antibody after i.v. bolus and after i.p. challenges suggest that TNF-alpha may not be as important in the pathogenesis of lethal peritonitis than after lethal acute bacteremia.     

Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice.

The systemic administration of human rIL-6 to mice resulted in the regression of established, 3-day pulmonary micrometastases from two weakly immunogenic tumors, but not from a nonimmunogenic tumor, in the absence of observable toxicity. Although IL-6 alone failed to have a significant therapeutic impact on advanced, 10-day pulmonary macrometastases from weakly immunogenic tumors, substantial cure rates of mice could be achieved when this cytokine was combined with cyclophosphamide. Histologic analysis of the lungs of mice receiving IL-6 revealed infiltration with lymphoid cells during the regression of pulmonary nodules from a weakly immunogenic tumor. IL-6-mediated tumor regression could be abrogated after selective in vivo depletion of either CD4 or CD8 T cell subsets by the systemic administration of specific mAb. In vivo generation of tumor-specific CTL, but not of lymphokine-activated killer cells, was detected in the lungs of IL-6-treated mice during regression of pulmonary metastases. Collectively, these findings demonstrate a role for IL-6 in the treatment of established solid tumors that have the capacity to elicit T cell responses in the host. Differences in host cellular mechanisms involved in tumor regression mediated by immunotherapy using IL-6 vs IL-2 are discussed.     

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.     

Seed and microsite limitation of recruitment in plant populations

Summary Availability of seed and microsites, respectively, are two factors that potentially may limit recruitment in plant populations. Microsites are small-scale sites suitable for germination and survival of seedlings. We discuss this dichotomy of recruitment limitation both from a theoretical and empirical point of view. Investigations of recruitment in 14 woodland species showed that 3 species were seed limited, 6 species were limited by a combination of seed and microsite availability, and 5 species were found not to be seed limited, but the limiting factor was not identified. A combination of seed and microsite limitation implies that recruitment is promoted by increasing both seed and microsite availability. We suggest that the importance of seed limitation in plant populations has been underestimated, and that the operating limiting factors may be dependent on spatial and temporal scale. We expect that many species, if adequately studied, will turn out to be both seed and microsite limited. Experimental field studies that incorporate a range of seed and microsite densities in various spatial and temporal scales are needed to examine the extent to which plant populations are seed and microsite limited.