A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.     

Comparative effects of four surfactants on growth,contraction and adhesion of cultured human fibroblasts

A range of surfactants, including the anionic sodium dodecyl sulfate, the cationic cetyltrimethylammonium bromide and the nonionics octylphenoxy polyethoxyethanol (Triton X100) and polyoxyethylene 20 sorbitan monoleate (Tween 80) was studied for effects on proliferation, contractibility and attachment of cultured human fibroblasts. Only ionic surfactants exhibited a stimulatory effect on fibroblast proliferation, whereas all the surfactants tested increased the contraction of collagen gels containing fibroblasts, with the greatest effect from the non-ionic surfactants. This activity was not correlated with an increase of cell population or cell attachment within the collagenous matrix. The activity of the surfactants was seen only at levels close to their LD₅₀ values and in a narrow range of concentrations. Thus, we consider that they are the result of the so-called hormesis phenomenon.Abbreviations CTAB cetyltrimethyl-ammonium bromide - FCS fetal calf serum - LD₁₀₀ dose lethal to 100% of exposed - MCD maximal contraction dose - PDL population doubling level - SDS sodium dodecyl sulfate     

Serum-free culture of primitive murine hemopoietic colony-forming cells

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.     

On the History of Men and Genocide

A historical and psychological study of genocide is presented in which special emphasis is given to modern manifestations of this phenomenon. The policy of eradication of the Jews by the Hitler regime is considered as an example of genocide in the twentieth century. The psychopathology of genocide and the complex relationships among aggressors, victims and witnesses are elaborated. Inferences are drawn from past and present patterns of genocide that may provide some leads to the future. It is now possible to envisage an all-consuming genocide unless nations can learn to live together, not necessarily in unity but in diversity.     

Influence of respiratory drive on upper airway resistance in normal men

The variations in nasal and pharyngeal resistance induced by changes in the central inspiratory drive were studied in 10 normal men. To calculate resistances we measured upper airway pressures with two low-bias flow catheters; one was placed at the tip of the epiglottis and the other in the posterior nasopharynx, and we measured flow with a Fleisch no. 3 pneumotachograph connected to a tightly fitting mask. Both resistances were obtained continuously during CO2 rebreathing (Read's method) and during the 2 min after a 1-min voluntary maximal hyperventilation. The inspiratory drive was estimated by measurements of inspiratory pressure generated at 0.1 s after the onset of inspiration (P0.1) and by the mean inspiratory flow (VT/TI). In each subject both resistances decreased during CO2 rebreathing; these decreases were correlated with the increase in P0.1. During the posthyperventilation period, ventilation fell below base line in seven subjects; this was accompanied by an increase in both nasal and pharyngeal resistances. These resistances increased exponentially as VT/TI decreased. Parallel changes in nasal and pharyngeal resistances were seen during CO2 stimulus and during the period after the hyperventilation. We conclude that 1) the indexes quantifying the inspiratory drive reflect the activation of nasopharyngeal dilator muscles (as assessed by the changes in upper airway resistance) and 2) both nasal and pharyngeal resistances are similarly influenced by changes in the respiratory drive.     

Marine macroalgae as a source for osmoprotection for Escherichia coli

At elevated osmolarity of the mineral medium M63, marine macroalgae constitute important osmoprotectants and nutrients sources for Escherichia coli. Growth of bacterial population (16 strains) was improved by supplementing M63 salts medium with either aqueous or ethanolic algal extracts obtained from Ascophyllum nodosum, Fucus serratus, Enteromorpha ramulosa, Ulva lactuca, and Palmaria palmata. In their presence, growth was still observed even at 1.02 m NaCl. Furthermore, the E. coli ZB400 growth in presence of whole macroalgae thalli in M63/0.85 m NaCI reached its maximum within 24 h (5 × 10⁷ – 5 × 10⁸ colony-forming units [CFU] per milliliter). In the presence of A. nodosum, bacterial growth was inhibited. In the same experimental conditions, ethanolic extracts improved E. coli growth significantly, because the yield reached 10¹¹ CFU per milliliter. Ulva lactuca and P. palmata allowed the better growth. The Dragendorff-positive compounds extracted from bacterial cells growing on each ethanolic extract exhibited an osmoprotective effect as proved by a disk-diffusion assay. On the other hand, the -onium compounds (quaternary ammonium [betaines] and tertiary sulphonium) and total free amino acid contents of U. lactuca ethanolic extracts were higher than in others. Fucaceae extracts demonstrated especially high protein content. Algal extracts constitute not only an appreciable osmoprotection source for E. coli but also nutrient sources. Correspondence to: J. Minet     

Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission

A 970 bp DNA fragment which encodes the luciferase enzyme of the marine soft coral Renilla reniformis was fused to the cauliflower mosaic virus (CaMV) promoter. The construct pPCV702-ruc was transferred into alfalfa protoplasts by Ca-PEG-mediated transformation and into tobacco, tomato and potato plants by Agrobacterium -mediated transformation. The light emission from homogenates of alfalfa protoplasts transformed with pPCV702-ruc was 16-fold higher than that of protoplasts transformed with the same vector carrying the bacterial luxF gene. Application of a 3 µM aequous solution of 2-benzyl luciferin (luciferin) on to calli, leaves, roots and slices of tomato fruits and potato tubers of transformed plants resulted in strong light emission within seconds which could be easily visualized by a photon counting camera. Light emissions obtained from tissue homogenates of tobacco plants containing a single copy of the pPCV702-ruc construct were around 20-fold higher than those from plants carrying multiple copies of the firefly luciferase gene and around 360-fold higher than those from plants transformed with the bacterial luciferase gene. Owing to its high efficiency the Renilla luciferase may become a useful and novel tool for gene expression studies in plants and other systems.