A small RNA that complements mutants in the RNA processing enzyme ribonuclease P

Four temperature-sensitive RNase P mutants were analyzed for the accumulation of 10 S RNA. In the 10 S region of the polyacrylamide gel two molecules appear, a and b. While the level of 10 Sa seems to be affected in some of the mutants, the 10 Sb molecule was not found in rnpB mutants. A plasmid (pL2), which contains Escherichia coli DNA sequences that complement, at least partially, rnp mutations, directs the synthesis of 10 Sb RNA. The presence of the pL2 plasmid complements the rnpA49, rnpB3187 and the rnpC241 mutations, as revealed by colony formation at “non-permissive” temperatures. However, the complementation of the rnpA49 mutation is much better than that of the other mutations. The complementation can also be measured by the increased level of RNase P activity in extracts. 10 Sa and b RNAs are unique among all RNAs tested thus far, since they are stable during exponential growth at 30 °C and 37 °C. However, at higher temperatures, such as 43 °C, the molecules are somewhat less stable, and they become rather labile when RNA synthesis is blocked by rifampicin. Structural analysis revealed that the 10 Sa and 10 Sb RNA molecules have dissimilar sequences.     

Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

Production of C3 as a marker of lymphokine-mediated macrophage activation

C3 production was assayed using an enzyme-linked immunosorbent assay (ELISA) in cell-free supernatants harvested from thioglycollate-elicited macrophages exposed to a variety of macrophage stimulating and activating agents. Macrophage monolayers treated with the stimulating agents starch, glycogen, and zymosan secreted three- to four-fold less C3 (mean 12 ng/10(5) cells/12 hr) than macrophages exposed to lymphokines containing macrophage-activating factor (MAF) (mean C3 production 44 ng/10(5) cells/12 hr). The increased production of C3 in macrophages exposed to MAF parallels the ability of these macrophages to acquire tumoricidal capacity as monitored in an in vitro 72 hr tumor cell cytotoxicity assay using B16 melanoma cells. Macrophages previously rendered tumoricidal by exposure to MAF and which are refractory to further challenge by MAF following decay of their tumoricidal properties, do not produce C3 on rechallenge with MAF. Exposure of refractory macrophages to liposome-encapsulated MAF overcomes the refractory state and induces re-expression of the tumoricidal phenotype and C3 production. We conclude that quantitative detection of macrophage-generated C3 antigen provides a useful biochemical marker for monitoring the acquisition of tumoricidal properties in macrophages exposed to MAF and offers a sensitive assay for screening novel agents that activate macrophages via mechanisms similar to MAF.     

Floral biology and pollinators of three co-occurring Cistus species (Gistaceae)

BOSCH, J., 1992. Floral biology and pollinators of three co-occurring Cistus species (Cistaceae). The pollination of three species of Cistus, Cistus albidus L., Cistus salvifolius L. and Cistus monspeliensis L. is studied. Insect visitors were censused and some were captured to analyse pollen distribution on their bodies. Floral phenology, breeding systems, some aspects of floral biology (pollen and nectar production, pollen-ovule ratios) and floral morphology were also studied for each species. Flowers of C. albidus produce more pollen and nectar than the other two species studied, and attract more insects, especially those with higher nutritional requirements. All three species are highly self-incompatible, but C. salvifolius and C. monspeliensis show some features that hinder xenogamy, and they have lower pollen-ovule ratios than C. albidus. As a result, this latter species apparently receives higher levels of cross-pollination, but at the cost of a higher investment to attract and reward reliable pollinators.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Fixed interval and fixed time treadle pressing in the pigeon: A comparison with FI and FT keypecking

Experiment I used non-naive pigeons having previously performed on both keypecking and treadlepressing Fixed Interval schedules. In condition IT, treadlepressing was reinforced on successive Fixed Interval 60 seconds, Fixed Time 60 seconds and Fixed Interval 60 seconds schedules. Subsequently (condition IK), the same subjects pecked a key on an identical schedule sequence (FI60, FT60, FI60). In Experiment II, separate groups of naïve subjects were assigned either to treadlepressing (condition IIT) or keypecking (condition IIK) and to the same schedule sequence (FI60, FT60, FI60). Treadle pressing and keypecking decreased greatly in Fixed Time schedules. Curvature indices, pauses and running rates were less sensitive than response rates to the switching from one schedule to the other. Experiments I and II yielded similar results, experimental history accounting only for minor differences. The results were discussed in relation to interspecies differences in the temporal regulation of behavior and operant versus respondent control of the response and schedule-induced behaviour.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Zn(II)-induced dimerization of human carbonmonoxy hemoglobin

Binding of Zn(II) to the carbon monoxide complex of human hemoglobin was shown by equilibrium sedimentation and sedimentation velocity experiments at pH 7.0 to induce the dissociation of liganded tetramers to dimers but not to monomers. These results provide direct confirmation of previous kinetic and gel filtration experiments (R. D. Gray, (1980) J. Biol. Chem.255, 1812–1818) that Zn(II) binding to liganded hemoglobin produces a change in aggregation state of liganded hemoglobin.     

MicroRNA-155 Regulates Cell Survival,Growth, and Chemosensitivity by Targeting FOXO3a in Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Despite improvement in treatment over the past few decades, there is an urgent need for development of targeted therapies. miR-155 (microRNA-155) is frequently up-regulated in breast cancer. In this study, we demonstrate the critical role of miR-155 in regulation of cell survival and chemosensitivity through down-regulation of FOXO3a in breast cancer. Ectopic expression of miR-155 induces cell survival and chemoresistance to multiple agents, whereas knockdown of miR-155 renders cells to apoptosis and enhances chemosensitivity. Further, we identified FOXO3a as a direct target of miR-155. Sustained overexpression of miR-155 resulted in repression of FOXO3a protein without changing mRNA levels, and knockdown of miR-155 increases FOXO3a. Introduction of FOXO3a cDNA lacking the 3′-untranslated region abrogates miR-155-induced cell survival and chemoresistance. Finally, inverse correlation between miR-155 and FOXO3a levels were observed in a panel of breast cancer cell lines and tumors. In conclusion, our study reveals a molecular link between miR-155 and FOXO3a and presents evidence that miR-155 is a critical therapeutic target in breast cancer.