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Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts 总被引:2,自引:0,他引:2
Luigi Atzori Jeannette M. Dypbukt Kristina Sundqvist Ian Cotgreave Charlotte C. Edman Peter Moldus Roland C. Grafstrm 《Journal of cellular physiology》1990,143(1):165-171
The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts. 相似文献
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Mirella Ruggeri Michele Zoli Roberta Grimaldi Urban Ungerstedt Agneta Eliasson Luigi F. Agnati Kjell Fuxe 《Neurochemistry international》1990,16(4):427-435
Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula. 相似文献
47.
The hydrolytic activity of lipolytic enzymes in reverse micelles can be measured continuously with Fourier Transform infrared spectroscopy (FTIR) by following in the region of the OH-stretching band the water consumption during the reaction. This possibility is unique to reverse micellar solutions, because they are optically transparent and because they contain only a limited amount of water. 相似文献
48.
Giovanni Fontana Roberto De Bernardi Federico Ferro Anita Gemignani Maurizio Raiteri 《Journal of neurochemistry》1996,66(1):161-168
Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg2+ -containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved. 相似文献
49.
Selenium,zinc, and thyroid hormones in healthy subjects 总被引:3,自引:0,他引:3
Oliviero Olivieri Domenico Girelli Anna Maria Stanzial Luigi Rossi Antonella Bassi Roberto Corrocher 《Biological trace element research》1996,51(1):31-41
Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T3 production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone
receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral
conversion of T4 to T3 with a lower T3/T4 ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating
and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully
selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects
were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample
of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T3/T4 ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation
between the T3/T4 ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as
compared with younger subjects. We concluded that reduced peripheral T4 conversion is related to impaired Se status in the elderly. 相似文献
50.
Stefania Canino Barbara Nieri Laura Pistelli Amedeo Alpi Luigi De Bellis 《Physiologia plantarum》1996,98(1):13-19
The activity of NADP+ -dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP+ -ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP+ , inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP+ -ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics. 相似文献