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31.
Borrell A Culianez-Macia FA Altabella T Besford RT Flores D Tiburcio AF 《Plant physiology》1995,109(3):771-776
Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. 相似文献
32.
Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis.
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We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis. 相似文献
33.
Cecilio CA Costa EH Ucelli P Chaves CA Toffoli MC Flores CA Cunha FQ Ferreira SH Tamashiro WM 《Mediators of inflammation》1997,6(1):46-52
Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites. 相似文献
34.
Several reports have claimed that the mitochondrial chaperonin cpn60, or a close homolog, is also present in some other subcellular compartments of the eukaryotic cell. Immunoelectron microscopy studies, using a polyclonal serum against cpn60, revealed that the protein is exclusively localized within the mitochondria of rat liver and embryonic Drosophila cells (SL2). Furthermore, no cpn60 immunoreactive material could be found within the nucleus of SL2 cells subjected to a 1 h 37°C heat-shock treatment. In contrast to these findings, immunoelectron microscopy studies, using a cpn60 monoclonal antibody, revealed mitochondrial and extramitochondrial (plasma membrane, nucleus) immunoreactive material in rat liver cells. Surprisingly, the monoclonal antibody also reacted with fixed proteins of the mature red blood cell. The monoclonal antibody, as well as cpn60 polyclonal sera, only recognize mitochondrial cpn60 in Western blots of liver proteins. Furthermore, none of the cpn60 antibodies used in this study recognized blotted proteins from rat red blood cells. Therefore, we suggest that the reported extramitochondrial localization of cpn60 in metazoan cells may be due to cross-reactivity of some of cpn60 antibodies with conformational epitopes also present in distantly related cpn60 protein homologs that are preserved during fixation procedures of the cells. © 1995 Wiley-Liss, Inc. 相似文献
35.
Whitening of Gracilaria chilensis, accompanied by tissue softening and thallus fragmentation, was found to be associated with the presence of an endophytic
amoeba. Although the symptoms developed originally in green mutant thalli, subsequent infections in the laboratory also affected
normal, wild-type G. chilensis. Ultrastructural evidence indicates that the amoebae perforate the host cell walls of both cortical and medullary cells and
digest their protoplasm. Feeding by the amoeba appears to involve both phagocytosis and enzymatic digestion of the host tissue.
Destruction of the host tissue resulted in large cavities first, followed by thallus fragmentation. No other organism was
found during the early stages of thallus invasion by the amoeba, although bacteria may appear once the amoeba reaches the
inner tissues of the host. 相似文献
36.
Osmotic Stress-Induced Polyamine Accumulation in Cereal Leaves : II. Relation to Amino Acid Pools 总被引:3,自引:2,他引:1
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Arginine decarboxylase activity increases 2- to 3-fold in osmotically stressed oat leaves in both light and dark, but putrescine accumulation in the dark is only one-third to one-half of that in light-stressed leaves. If arginine or ornithine are supplied to dark-stressed leaves, putrescine rises to levels comparable to those obtained by incubation under light. Thus, precursor amino acid availability is limiting to the stress response. Amino acid levels change rapidly upon osmotic treatment; notably, glutamic acid decreases with a corresponding rise in glutamine. Difluoromethylarginine (0.01-0.1 millimolar), the enzyme-activated irreversible inhibitor of arginine decarboxylase, prevents the stress-induced putrescine rise, as well as the incorporation of label from [14C]arginine, with the expected accumulation of free arginine, but has no effect on the rest of the amino acid pool. The use of specific inhibitors such as α-difluoromethylarginine is suggested as probes for the physiological significance of stress responses by plant cells. 相似文献
37.
38.
A. G. Fazekas J. Marti M. Flores J. K. MacFarlane D. M. P. Thomson 《Cancer immunology, immunotherapy : CII》1978,5(2):145-147
Summary The intensity of cortisol binding was measured in the cytosol fraction of the primary tumor obtained from 50 patients with stage I and II breast cancer. The state of cellular antitumor immunity of the same patients was investigated by the tube leucocyte adherence inhibition (LAI) test, performed with peripheral blood leucocytes 1–2 days preoperatively. It was found that the intensity of tumor cortisol binding correlates negatively with LAI values. Patients with high cortisol binding in their tumors have low LAI values, while low tumor cortisol binding is associated with higher antitumor immunity. The results suggest that high cortisol binding in the tumor might inhibit the tumor recognition process and/or the cellular immune defense mechanism and thus facilitate cancer development. 相似文献
39.
Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.
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J Fischer T R Kohler L G Lipson J Flores P A Witkum G W Sharp 《The Biochemical journal》1978,173(1):59-64
Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site. 相似文献
40.