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171.
We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed. 相似文献
172.
María del Carmen García Manuela G. López Antonio G. García Mariano Sánchez Crespo 《Journal of neurochemistry》1992,59(6):2244-2250
Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor. 相似文献
173.
Bonaventura Ruiz-Montasell F. Javier Casado Antonio Felipe Marçal Pastor-Anglada 《The Journal of membrane biology》1992,128(3):227-233
Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K
M 8.9
m and V
max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na+ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na+ counterions determined uridine transport rates (SCN– > NO
3
–
> Cl– > SO
4
2–
). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme. 相似文献
174.
Raúl Alva J. Antonio Lugo-R Eduardo Arzt Jorge Cerbón Blanca E. Rivera Mireya Toro Sergio Estrada-O 《Journal of bioenergetics and biomembranes》1992,24(1):125-129
Nigericin is a monocarboxylic polyether molecule described as a mobile K+ ionophore unable to transport Li+ and Cs+ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li+, Cs+, Na+, Rb+, and K+. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li+ and Cs+ than for Na+, K+, and Rb+, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li+ and nigericin-Cs+ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na+, K+, and Rb+ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li+ and Cs+ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K+>Rb+>Na+.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia. 相似文献
175.
176.
Motoshige Kawata Satoshi Harada Baltazar Antonio Kiyoharu Oono 《Plant Cell, Tissue and Organ Culture》1992,28(1):1-10
Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants. 相似文献
177.
Mario Díaz Antonio Lorenzo 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(2):189-196
Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I
sc), as well as in the mucosa-to-serosa (J
m-s
Na
) and net sodium flux (J
net
Na
). In AT tissues, aldosterone did not change net chloride flux (J
net
Cl
) but did so in CT tissues. Amiloride reduced the aldosterone-increased I
sc in AT and CT tissues, inhibited J
net
Na
in AT tissues and abolished it in CT colons. J
net
Cl
was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na+ absorption and totally inhibited Cl- absorption in the AT tissues, but did not change I
sc. However, in CT tissues neither Na+ nor Cl- transport were affected by DIDS. A good relationship between I
sc and J
m-s
Na
was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J
net
Na
and reduced I
sc to untreated control values. Addition of serosal ouabain abolished I
sc and Na+ absorption in AT and CT colons, but Cl- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT
acutely treated
- CT
chronically treated animals
- DIDS
4-4-diisothiocyanatostibene-2-2-disulfonic acid
- DMSO
dimethylsulphoxide
-
G
t
tissue conductance
-
I
sc
short circuit current
- PD
transepithelial potential difference
- SITS
4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid
- UC
untreated controls
Preliminary results of this paper were presented at the X
th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990 相似文献
178.
Manuel Mari-Beffa José F. de Celis Antonio García-Bellido 《Development genes and evolution》1991,200(3):132-142
Summary The role of the achaete-scute complex and extramacrochaetae, Notch, Delta, Enhancer of split and Hairless genes in chaeta patterning in Drosophila tergites was studied in genetic mosaics and in mutant combinations. The mutant phenotypes of different alleles of each gene can be ordered in characteristic topographical seriations. These seriations are related to the pattern of proliferation of histoblasts and the time of singularization of sensory organ mother cells from surrounding epidermal cells. Genetic mosaics of lethal alleles show that these genes are fundamentally involved in this singularization and subsequent differentiation. The study of mutant combinations of alleles of these genes reveals specific relationships of epistasis and synergism between them. The results suggest that spatial and temporal variations in achaete-scute complex functional products in cells, modulated by the activity of other genes involved in signal transduction, define the patterned differentiation of sensory organs in tergites.
Offprint requests to: A. García-Bellido 相似文献
179.
180.