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41.
Kost Benedikt; Galli Alessandro; Potrykus Ingo; Neuhaus Gunther 《Journal of experimental botany》1995,46(9):1157-1167
An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into2040 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml1or 1 mg ml1 have been tested. With 1 mg ml1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation 相似文献
42.
Alessandro Sisto Fabio Bonelli Felice Centini Christopher I. Fincham Edoardo Potier Edith Monteagudo Paolo Lombardi Federico Arcamone Cristina Goso Stefano Manzini Alessandro Giolitti Carlo A. Maggi Mariano Venanzi Basilio Pispisa 《Biopolymers》1995,36(4):511-524
In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc. 相似文献
43.
Alessandro Terrinoni Carmen Di Franco Patrizio Dimitri Nikolaj Junakovic 《Journal of molecular evolution》1997,45(2):145-153
The intragenomic location of the elements of the I, G, jockey, F, and Doc transposon families has been studied by the Southern
blot analysis, in 12 laboratory Drosophila melanogaster stocks. Elements located in euchromatin, heterochromatin, and on the Y chromosome are identified, and their stability has
been assessed by comparing the autoradiographs detected in different stocks and analysis of individual flies. Evidence is
shown suggesting that preferential location in euchromatin or heterochromatin and the distribution within heterochromatin
are distinctive of transposon families. Elements located in heterochromatin can be unstable. These results are discussed in
the context of the relationship between transposable elements and the host genome.
Received: 21 August 1996 / Accepted: 24 March 1997 相似文献
44.
†Alessandro P. Burlina †Vanni Ferrari ‡Laura Facci ‡Stephen D. Skaper § Alberto B. Burlina 《Journal of neurochemistry》1997,69(3):1314-1317
Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve-mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide-containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N -acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease. 相似文献
45.
Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry 总被引:3,自引:0,他引:3
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Paolo Macchi Anna Villa Dario Strina Maria Grazia Sacco Federica Morali Duilio Brugnoni Silvia Giliani Elide Mantuano Anders Fasth Bengt Andersson Ben J. M. Zegers Giovanni Cavagni Igor Reznick Jacov Levy Israel Zan-Bar Yael Porat Paolo Air Alessandro Plebani Paolo Vezzoni Luigi D. Notarangelo 《American journal of human genetics》1995,56(4):898-906
X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR. 相似文献
46.
Macarena Gomez-Lira Antonella Sangalli Monica Mottes Chiara Perusi Pier Franco Pignatti Nicolò Rizzuto Alessandro Salviati 《Human genetics》1995,96(4):417-422
-Hexosaminidase gene mutations were analyzed in two adult-onset Sandhoff disease Italian patients by PCR analysis of a common known mutation (5) and by heteroduplex analysis of genomic and RT-PCR DNA fragments, covering the whole gene. The patients' genotypes were 5/C1214T, and G890A/C1214T, respectively. As mutation C1214T (Pro405Leu) is also present in the other two late-onset cases so far described, we suggest that C1214T is a common mutation in this type of Sandhoff disease. Mutation G890A (Cys297Tyr) is a novel mutation which presumably causes altered processing of the pro chain. 相似文献
47.
The basic anatomy of lateral twig insertion onto the main branch in both healthy and damaged Quercus cerris L. trees was studied. An abscission zone is always present: in healthy trees it is formed by a smaller number of cell layers than in damaged ones, where it is more evident with many layers of cells. Cells of the abscission zone are roundish, with many intercellular spaces between them; cell walls are thin, non-lignified and without secondary walls. No starch was found in cells of the abscission zone, where, instead, a few scattered calcium oxalate druses are seen. 相似文献
48.
Alessandro Ferrari Felice Petraglia Erlio Gurpide 《The Journal of steroid biochemistry and molecular biology》1995,54(5-6):251-255
Decidualization of endometrial cells is a hormone-dependent process of differentiation which occurs during the menstrual cycle and pregnancy. Recent in vitro studies have revealed that cAMP and its generators induce decidualization of stromal cells isolated from proliferative endometrium and that progestins enhance the effect of cAMP. Since corticotropin releasing factor (CRF) generates cAMP and prostaglandins in other organs, in the present study the effect of CRF, a hypothalamic factor also produced by decidua and fetal membranes, on in vitro decidualization of endometrial stromal cells was evaluated. The addition of CRF to a culture medium of stromal cells induced in vitro decidualization, as indicated by morphologic changes from elongated fibroblast-like cells into larger and round cells and by the release of prolactin in the medium. The effect of CRF on stromal cells and on prolactin release was significantly augmented by the coincubation in the presence of medroxyprogesterone acetate. This observation indicates CRF as a novel factor of decidualization and confirms that progestins act as enhancers of the expression of decidual products. 相似文献
49.
Leilo Zolla Harry A. Kuiper Alessandro Finazzi Agrò Maurizio Brunori 《Journal of inorganic biochemistry》1984,22(3):143-153
Addition of KCN to Helix pomatia β-hemocyanin fully saturated with either O2 or CO results in a decrease of the spectroscopic properties of the protein (absorbance at 340 nm and luminescence at 550 nm) due to the displacement of the gaseous ligands (O2 or CO) from the active site. The anionic form of cyanide (CN?) is supposed to bind to the active site; its intrinsic affinity for the protein, as calculated from independent O2 and CO displacement experiments, is between 2 and 6 × 106M?1. The replacement of O2 or CO shows some differences which may be correlated with the different modes of binding at the active site. Thus, while displacement of oxygen by cyanide is hyperbolic, addition of cyanide to carbonylated hemocyanin shows a lag phase. This finding suggests the formation of a mixed liganded complex at the active site. The simultaneous presence of CO and CN? at the active site of hemocyanin is also supported by the experiment in which addition of small amounts of KCN to hemocyanin partially saturated with O2 and CO gives rise to an increase of emission intensity and a concomitant decrease of the O2 absorption band. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation. 相似文献
50.
Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [3H]glucosamine, [3H]mannose or [3H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.Abbreviations endo H
endo--N-acetylglucosaminidase H
- GlcN
glucosamine
- GlcNAc
N-acetylglucosamine
- Man
mannose
- PHA
phytohemagglutinin
This work was done while A.V. was on leave from the Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, I-20133 Milano, Italy 相似文献