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991.
The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuro-blastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [3H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [3H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPS. The stimulation of [3H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei. 相似文献
992.
993.
Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k) 总被引:1,自引:0,他引:1
A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO3
− metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones.
The final screening was performed using NO3
−, NO2
− or NH+
4 as N-source. The mutants isolated absorb NO3
− with rates close to those measured for the wild type and they excrete NO2
− into the medium. The ratio between NO3
− uptake and NO2
− excretion was 1:1. The sensitivity of NO3
− uptake to NH+
4 was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback
regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of
the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising
tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling
of C and N metabolism by inorganic N-compounds.
Received: 8 October 1999 / Accepted: 25 January 2000 相似文献
994.
Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT)
were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO2 assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression
of the endogenous TPT (αTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO2, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or
CO2 assimilation. However, in elevated CO2 (1500 μl · l−1) and low O2 (2%) the TPT exerted strong control on the rate of CO2 assimilation (control coefficient for the wild type; CJA
TPT=0.30) in saturating light. Similarly, the incorporation of 14C into starch in high CO2 was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a CJStarch
TPT=−0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control
strength on the rate of sucrose biosynthesis (CJSuc
TPT=0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical
to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration
showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in αTPT plants with
an apparent control coefficient of CJRes
TPT=0.24. If the control on sucrose biosynthesis is referred to as “gain of carbon” (positive control) and the control on starch
biosynthesis as well as dark respiration as a “loss of carbon” (negative control) for sucrose biosynthesis and subsequent
export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the
control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron
transport, but only at high light and in elevated CO2 combined with low O2. The control coefficient for the rate of photosynthetic electron transport was CJETR
TPT=0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore,
the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed.
The TPT also exerted control on metabolite contents in air.
Received: 26 March 1999 / Accepted: 21 August 1999 相似文献
995.
In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects. 相似文献
996.
Mesnard F Azaroual N Marty D Fliniaux MA Robins RJ Vermeersch G Monti JP 《Planta》2000,210(3):446-453
Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (15NH4)2SO4 and K15NO3. By using two-dimensional 15N-1H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences,
an enhanced resolution of the incorporation of 15N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional
15N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore,
it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional 15N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of 15N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate
to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and
of the nucleotide uridine could be seen.
Received: 29 March 1999 / Accepted: 15 July 1999 相似文献
997.
Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons. 相似文献
998.
Adenosine is a neuromodulator, and it has been suggested that cerebral acetate metabolism induces adenosine formation. In the present study the effects that acetate has on cerebral intermediary metabolism, compared with those of glucose, were studied using the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Fasted rats received an intravenous injection of CCPA, DPCPX, or vehicle. Fifteen minutes later either [1,2-13C]acetate or [1-13C]glucose was given intraperitoneally; after another 30 min the rats were decapitated. Cortical extracts were analyzed with 13C NMR spectroscopy and HPLC analysis. DPCPX affected neuronal and astrocytic metabolism. De novo synthesis of GABA from neuronal and astrocytic precursors was significantly reduced. De novo syntheses of glutamate and aspartate were at control levels, but their degradation was significantly elevated. In glutamine the anaplerotic activity and the amount of label in the position representing the second turn in the tricarboxylic acid cycle were significantly increased, suggesting elevated metabolic activity in astrocytes. CCPA did not influence GABA, aspartate, or glutamine synthesis. In glutamate the contribution from the astrocytic anaplerotic pathway was significantly decreased. In the present study the findings in the [1,2-13C]acetate and [1-13C]glucose control, CCPA, and DPCPX groups were complementary, and no adenosine A1 agonist effects arising from cerebral acetate metabolism were detected. 相似文献
999.
The inhibitory or antibiotic action of four kinds of the selenomorpholine complex on a strain of Escherichia coli was studied by microcalorimetry. Differences in their capacities to inhibit the metabolism of this bacterium were observed.
The extent and duration of the inhibitory effect on the metabolism as judged from the rate constant, k, and the half-inhibitory concentration, IC50, varied with the different drugs. The rate constant (k) of Escherichia coli (in the log phase) in the presence of the drugs decreased with increasing concentrations of the drugs (C). The relationship of k and C is nearly linear for (1) selenomorpholine and (2) selenomorpholine hydrochloride, but for (3) N,N′-methylene bisselenomorpholine and (4) N-dodecyl selenomorpholine, it is not linear. The experimental results reveal that the sequence of antibiotic activity of selenomorpholines
is (3) and (4)>(1)>(2). 相似文献
1000.