Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Actin stringently accumulated in the specifically positioned,differentiating female gametangia of Allomyces

Summary Female gametangia of the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent of their apical or subapical positioning during differentiation on the fertile hyphae. The anti-actin, cytochalasin D, can selectively suppress male differentiation in both species.     

Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer

Of the total number of breast cancers approx. 30-50% are hormone-dependent and estradiol is one of the main factors of cancerization. Consequently, the control of this hormone inside the cancer cell is of capital importance because it is well established that the inhibition of estradiol biosynthesis can have a positive effect on the evolution of the disease. The blockage of estradiol can be obtained by the action of anti-aromatases, anti-sulfatases, the control of the 17 beta-hydroxysteroid dehydrogenase activity or by the stimulation of the sulfotransferase which converted the estrogens in their sulfates. In breast cancer tissue estrone sulfate is quantitatively the most important source of estradiol. In the intact cell, estrone sulfatase activity is very intense in the hormone-dependent cell lines (e.g. MCF-7, T-47D) but very small activity is observed in the hormone-independent (e.g. MDA-MB-231, MDA-MB-436) cell lines. However, this activity became very strong after homogenization in the hormone-independent cells, suggesting the presence of repressive factor(s) for this enzyme or its sequestering in an inactive form, in the intact cells of these cell lines. In a series of previous studies it was found that in hormone-dependent cell lines different anti-estrogens: tamoxifen and derivatives, ICI 164,384, very significantly decrease the estradiol concentration originated from estrone sulfate, and recently it was observed that Decapeptyl (D-Trp6-gonadotropin-releasing hormone) in the presence of heparin can also decrease the conversion of estrone sulfate into estradiol. No significant effect was obtained in the presence of heparin or Decapeptyl alone. The estrone sulfatase activity can be inhibited by progesterone, the progestagen R-5020, and testosterone. In another series of recent studies the presence of very strong estrogen sulfotransferase activity has been shown in one breast cancer cell line, the MDA-MB-468. We can conclude that: (1) the control of estradiol concentration can be carried out in the breast cancer tissue itself; (2) estrone sulfate can play an important role in the bioavailability of estradiol in the breast cancer cell; and (3) as is the case for the aromatase, the control of: the estrogen sulfatase, estrogen sulfotransferase, and 17 beta-hydroxysteroid dehydrogenase can be new targets for therapeutic applications in breast cancer.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.     

Conformational analysis and molecular dynamics simulations of maltose

Constrained conformational energy minimizations have been used to calculate an adiabatic (Φ, ψ) potential energy surface for the disaccharide β-maltose. The inclusion of molecular flexibility in the conformational energy analysis of the disaccharide was found to significantly lower the barriers to conformational transitions, as has been observed previously for other systems. Several low energy wells were identified on the adiabatic surface which differ in energy by small amounts and with low absolute barriers separating them, indicating the possibility of a non-negligible equilibrium population distribution in each well. If such a distribution of conformations existed in the physical system, the conformation observed by NMR NOE measurements would thus be a “virtual” conformation. Molecular dynamics simulations of the motions of this molecule in vacuum were also conducted and indicate that the rate of relaxation of the molecule to the adiabatic surface may be slower than the typical timescale of conformational fluctuations. This effect is apparently due to an unphysical persistence of hydrogen bond patterns in vacuum which does not occur in aqueous solution. Trajectories undergoing transitions between wells were calculated and the effects of such conformational transitions upon the ensemble mean structure, such as might be observed in an NMR experiment, were demonstrated.