3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: A new fluorescent dye to detect mycoplasma contamination in cell cultures

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

T-DNA length variability in mannopine hairy root: more than 50 kilobasepairs of pRi T-DNA can integrate in plant cells

Independent carrot (Daucus carota) hairy root lines were established by inoculation of discs taken from the same carrot with Agrobacterium rhizogenes 8196 and A. tumefaciens C58C1(pRi8196) carrying pRi8196. Several lines were compared with respect to T-DNA length. One of them was found to have integrated sequences covering more than 50 kbp of the Ri plasmid     

Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases

Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, paranitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.Abbreviations EGF Epidermal Growth Factor - IGF I Insulin-like Growth Factor I - PDGF Platelet-Derived Growth Factor - DMEM Dulbecco's Modified Eagle's Medium - FCS Fetal Calf Serum - PBS Phosphate Buffered Saline - PNPP Para-nitrophenylphosphate - BSA Bovine Serum Albumin - -Tyr Antiphosphotyrosine Antibodies - MAP 2 Microtubule-Associated Protein 2 - Hepes N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - EDTA Ethylenediamine Tetraacetic Acid - DTT Dithiothreitol - SDS-PAGE Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis - EGTA [Ethylenebis(oxyethylenenitrilo)] Tetraacetic Acid - TRIS Tris(hydroxymethyl)-Aminoethane - IRSK Insulin Receptor-Associated Serine Kinase - KIK Kemptide Insulin-stimulated Kinase     

Physiological characterization of opine-utilizing rhizobacteria for traits related to plant growth-promoting activity

Thirty-two strains of opine-utilizing rhizobacteria were evaluated for physiological traits which have been related to plant growth-promoting activity. Tests included antibiosis against two bacterial and eight fungal pathogens of potato (Solanum tuberosum L.), production of hydrogen cyanide and fluorescent pigment production. On average, 71 and 12% of the bacteria inhibited the growth of Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively. The growth of Botrytis sp. was inhibited by 62% of the bacteria, and half of these produced an inhibition zone of more than 7 mm in diameter. Fusarium solani, Colletotrichum coccodes, Phoma exigua, Verticillium dahliae, F. oxysporum, V. albo-atrum and F. sambucinum were antagonized by 43, 34, 31, 25, 19, 18, and 12% of the bacteria, respectively. Only four strains produce hydrogen cyanide. The inhibition of a plant pathogen was not correlated to the production of fluorescent pigment. No strain produced a hypersensitive reaction whereas only three strains induced soft-rot and two produced polygalacturonase. Some opine-utilizing rhizobacteria were strong inhibitors of all plant pathogens, while most were active against specific plant pathogens.     

Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5

Transgenic tomato plants expressing antisense RNA to a ripening-related cDNA clone (pTOM5) had yellow ripening fruit and pale coloured flowers. Carotenoid levels in fruit of these plants were reduced by up to 97%. In order to determine the step of carotenoid biosynthesis which was blocked, a cell-free system active in the synthesis of carotenoid intermediates was prepared. Incubations with radiolabelled carotenoid precursors led to the identification of the block between GGDP and phytoene. Analysis of carotenoids in different tissues of transgenic and control plants indicated that although ripe fruit and flower carotenoid levels were reduced in the modified plants, leaf carotenoid levels were not decreased. This implies that the pTOM5 gene product is not involved in carotenoid synthesis in the leaf.     

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.     

Isolation of low-copy-number sequences that neighbor satellite DNA in mammals

To investigate the role of satellite DNA in eukaryotic genomes, we isolated from an African green monkey (Cercopithecus aethiops) genomic library cloned segments containing the previously described deca-satellite linked to low-copy-number genomic sequences. Three such clones were obtained. The low-copy-number sequences in the three clones do not cross-hybridize suggesting that they derive from different genomic loci. The structure of one of the clones, λAMkA, is described in detail. Subcloned segments containing the low-copy-number sequences from λAMkA anneal to monkey, human and mouse genomic DNA. The subcloned probes were used to select clones containing homologous sequences from a second, independent monkey library as well as from human and mouse genomic libraries. Several of the newly isolated monkey clones hybridized to probes containing the species-specific deca- and -satellites, confirming the genomic association of the low-copy-number sequence in λAMkA with satellite DNA. Moreover, several of the human and mouse clones hybridized to species-specific human and mouse satellite DNAs, respectively. These experiments indicate that the low-copy-number sequence in λMkA and its association with satellite DNA is conserved in primates and rodents.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid