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1.
The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the Km and Vmax of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca2+, Mg2+, K+, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca2+, K+, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca2+, K+, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca2+, K+, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients. 相似文献
2.
The principles of Henri-Michaelis-Menten kinetic analysis were applied to directly relate the concentration of serum growth factors and individual nutrients in the culture medium to the multiplication rate of a population of normal (N-HLF) and SV40 virus-transformed (SV-HLF) human lung fibroblasts. When all nutrient concentrations were optimal and in steady state, the concentration of serum factors that was required to support a half-maximal rate of proliferation of both N-HLF and SV-HLF was similar. When the serum factor concentration was optimal and constant, SV-HLF cells exhibited a reduced requirement (p less than 0.001) for 12 of 27 individual nutrients that were examined. Serum factors control the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of N-HLF (McKeehan, W. L., and McKeehan, K. A. (1980) Proc. Natl, Acad, Sci, U. S. A. 77, 3417-3421). SV-HLF exhibited a constitutively reduced requirement for Ca2+, K+, and Mg2+ which partially removed the requirement for the 3 ions for multiplication of SV-HLF from the control of serum factors. The results suggest that SV40 virus transformation confers a growth advantage on human lung fibroblasts by alteration of their quantitative requirements for specific nutrients. 相似文献
3.
《Journal of Fermentation Technology》1986,64(2):109-117
An organism isolated from soil by enrichment on isonicotinic acid (INA) was characterized as Bacillus brevis. It used sugars more readily than amino acids as growth substrates. The organism also used isoniazid, 2-hydorxypyridine, and benzoic and p-hydroxybenzoic acids. This bacterium did not metabolize 2-hydroxy-INA, citrazinic acid, or other mono- and dihydroxypyridine compounds as well as intermediates of the maleamate pathway. Accumulation of hydroxylated pyridine compounds was not detected during fermentation, or incubation of INA with resting cells in the presence or absence of inhibitors. Succinic semialdehyde was isolated and characterized as a key intermediate and was rapidly oxidized by INA-adapted cells. Formate was detected as a product of INA metabolism, and formate but not formamide was oxidized by INA-adapted cells; γ-aminobutyrate or γ-aminocrotonate were oxidized. A pathway for INA degradation involving oxygenative cleavage of a partially reduced pyridine ring is proposed. 相似文献
4.
M S Risley 《Biology of reproduction》1990,42(3):511-522
The support of Xenopus laevis spermatogenesis in vitro by different energy-yielding substrates has been investigated. Isolated spermatogenic cells maintained their levels of adenosine-triphosphate for 24 h in serum-free medium containing only amino acids as energy substrates. DL-Aminocarnitine, an inhibitor of carnitine palmitoyltransferase, reduced cell viability 87% during a 15-h culture in the same medium, indicating that beta oxidation of endogenous fatty acids is a significant source of energy when exogenous substrates are unavailable. Isolated spermatocytes developed into spermatids for 7 days in medium supplemented with either pyruvate, oxaloacetate, or lactate, with maximal survival and development at 0.5 mM pyruvate, 2.0 mM oxaloacetate, and 4.0 mM lactate. Few spermatocytes survived more than 3 days in serum-free medium supplemented with only glucose and amino acids as energy substrates. In contrast, glucose-supplemented medium supported spermatocyte differentiation for 14 days in testis fragment culture and 7 days in spermatocyte-Sertoli cell cocultures due to the excretion of lactate and pyruvate by Xenopus Sertoli cells during culture in glucose-supplemented medium. Glucose also enhanced spermatocyte development in medium containing dialyzed, heat-inactivated fetal calf serum. Spermatogenic cells oxidized glucose to CO2 with C1 oxidized 6- to 7-fold more than C6, suggesting that glucose may be metabolized in the hexose monophosphate shunt. The results are discussed in comparison to energy metabolism in mammalian testes and spermatogenic cells. 相似文献
5.
Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart 总被引:3,自引:1,他引:2
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In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart. 相似文献
6.
Alkylhydroperoxide reductases (AhpR, EC 1.6.4.*) are essential for the oxygen tolerance of aerobic organisms by converting otherwise toxic hydroperoxides of lipids or nucleic acids to the corresponding alcohols. The AhpF component belongs to the family of pyridine nucleotide-disulphide oxidoreductases and channels electrons from NAD(P)H towards the AhpC component which finally reduces cognate substrates. The structure of the catalytic core of the Escherichia coli AhpF (A212-A521) with a bound FAD cofactor was determined at 1.9 A resolution in its oxidized state. The dimeric arrangement of the AhpF catalytic core and the predicted interaction mode between the N-terminal PDO-like domain and the NADPH domain favours an intramolecular electron transfer between the two redox-active disulphide centres of AhpF. 相似文献
7.
Akimov MG Gretskaia NM Shevchenko KV Shevchenko VP Miasoedov NF Bobrov MIu Bezuglov VV 《Bioorganicheskaia khimiia》2007,33(6):648-652
Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time. 相似文献
8.
To assess the disulfide reduction capacity of intact cells, EA.hy926 endothelial cells were incubated with alpha-lipoic acid in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Alpha-lipoic acid was reduced within cells to dihydrolipoic acid, which could be quantified upon efflux from the cells as reduction of DTNB. Uptake of both alpha-lipoic acid and alpha-lipoamide occurred at least in part via a medium chain fatty acid transporter, based on inhibition by octanoate. Alpha-lipoic acid was reduced within cells by pyridine nucleotide-disulfide oxidoreductases, since it is not reduced by GSH and since its reduction was inhibited by carmustine. Nonetheless, reduction was also dependent on the cellular redox environment, since it was inhibited by the redox cycling of menadione, by decreasing intracellular GSH, and by reduction of dehydroascorbate. Together, these results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to assess the disulfide-reducing capacity of intact cells, especially as determined by pyridine nucleotide-disulfide oxidoreductases. 相似文献
9.
H.M. Liebich 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1983,273(1):67-75
Total 4-heptanone is the sum of a β-oxocarboxylic acid, 2-ethyl-3oxohexanoic acid, and its decarboxylation product, 4-heptanone.The β-oxocarboxylic acid is found in serum and in urine, and is detected by gas chromatography-mass spectrometry in the form of its methyl ester or its O-methyloximated acid methyl ester. The ketone is detected within the profile of volatile metabolites in serum and in urine. However, the analytical procedure includes some ketone coming from in vitro decarboxylation.Total 4-heptanone is measured by gas chromatography-mass fragmentography. The method includes a quantitative transformation of the β-oxocarboxylic acid into the ketone. A comparative study with 270 patients with diabetes mellitus, 28 healthy individuals and 143 non-diabetic hospitalized patients showed that the urinary excretion of total 4-heptanone is increased in diabetes mellitus. The mean values are 1073 μg per 24 h for diabetics, compared with 207 μg per 24 h and 246 μg per 24 h for healthy individuals and non- diabetic patients, respectively. Diabetic ketoacidosis and fasting conditions decrease the total 4-heptanone. 相似文献
10.
Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors. 相似文献
11.
When embryonic central nervous system neurons are seeded at low densities with Eagle's basal medium supplemented with the serum substitute N1, glucose, and glutamine, neuronal survival for even 24 h requires the additional supply of exogenous pyruvate--and so does the survival of many peripheral nervous system neurons. Pyruvate can be replaced by alpha-ketoglutarate or oxaloacetate, but not by Krebs cycle substrates that are not keto acids. Most other alpha-keto acids tested (though not beta- or gamma-keto acids) also mimic pyruvate. The apparent equivalence to pyruvate of all these compounds includes identical ED50 values (300 microM for embryonic avian fore-brain neurons, 30-40 microM for rat hippocampal neurons), and also identical susceptibilities to the pyruvate-sparing effects of other low-molecular-weight agents present in Dulbecco's modified Eagle's medium or in astroglia conditioned medium. The substitute alpha-keto acids, however--unlike pyruvate, alpha-ketoglutarate, or oxaloacetate--support cell survival only in the presence of alpha-amino acids that transaminate to alpha-ketoglutarate, oxaloacetate, or pyruvate. The alpha-keto acids, therefore, operate as acceptors of amino groups from appropriate donors to generate Krebs cycle-relevant substrates. Consistent with this view, [14C]glutamate did not generate appreciable 14CO2 unless accompanied by a suitable alpha-keto acid.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
Control of normal and transformed cell proliferation by growth factor-nutrient interactions 总被引:1,自引:0,他引:1
W L McKeehan 《Federation proceedings》1984,43(1):113-115
The proliferation of normal mammalian cells, similar to that of single bacterial and lower eukaryotic cells, is restricted by space and nutrients. Cultured human lung fibroblasts have been used as a model to show that, in the absence of spatial restrictions, the requirement for specific nutrients limits normal cell proliferation. Serum-derived hormonelike growth factors transiently reduce the requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for normal cell proliferation. Oncogenic transformation by virus causes a constitutive reduction in the requirement for multiple nutrients for proliferation. A constitutive reduction in the proliferative requirement for Ca2+, K+, and Mg2+ allows transformed cells to escape the restrictions imposed on normal cell growth by suboptimal external concentrations of Ca2+, K+, Mg2+, and hormonelike growth factors. An understanding of the processes that determine the nutrient requirements of different normal cell types and their alteration by hormonelike growth factors and oncogenic agents is needed to understand and suppress the growth advantage possessed by malignant cells. 相似文献
13.
Microbial Decomposition of alpha-Picoline 总被引:3,自引:0,他引:3
O P Shukla 《Indian journal of biochemistry & biophysics》1974,11(3):192-200
An organism, which degrades alpha-picoline but also utilizes 2-ethylpyridine or piperidine as alternative growth substrates, has been isolated from soil and characterized as arthrobacter sp. alpha-picoline-grown cells oxidize 2-ethylpyridine and vice versa. Other pyridine derivatives tested are neither utilized as growth substrates nor oxidized by the organism. alpha-Picolinate and 2-hydroxy-6-methylpyridine are not metabolized, indicating that degradation is neither initiated by methyl oxidation nor by hydroxylation in the 6-position of pyridine ring. Succinate semi-aldehyde and pyruvate accumulate when alpha-picoline oxidation by resting cell suspensions is blocked by semicarbazide. The Arthrobacter grown on alpha-picoline rapidly oxidizes succinate semi aldehyde... 相似文献
14.
Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells 总被引:4,自引:0,他引:4
These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation. 相似文献
15.
Lactic acid production by chick embryo fibroblasts occurs in the absence of exogenous glucose. Fifteen to 50-fold less lactic acid is formed in the absence of glucose than in its presence. Nevertheless, serum and pH stimulation enhances this residual lactic acid production to the same relative extent as when glucose is present. The amount of lactic acid formed cannot be accounted for by the catabolism of residual glucose in the medium since its concentration is less than one-tenth that of the lactic acid eventually produced. Moreover, the residual glucose concentration remains constant or increases during the course of the experiment. To a large extent lactic acid accumulation in the absence of external glucose is dependent on the presence of amino acids in the medium, but amino acid transport is not affected by the stimulatory agents used in this study. The results suggest that treatments which stimulate cell multiplication also activate those enzymatic pathways which convert amino acids to pyruvic and thence to lactic acid. 相似文献
16.
G Zaccone 《Basic and applied histochemistry》1984,28(1):29-40
The activities of various oxidative enzymes and the presence of acid carbohydrates and acid glycoproteins in the epithelial cells and the mucous goblet cells of the epidermis of the Teleost Electrophorus electricus have been determined by means of a series of selected histochemical techniques of light microscopy. The enzyme activities show a distribution pattern confined mainly to basal cell layers and outer cell layers with a comparatively lower gradient in the transitional cell layers. A mixture of sialic acids of both N-acylated and O-acylated types is found in the mucous goblet cells and the functional significance of mucous production is related to the first line of defense against pathogenic colonization. The higher incidence of various oxidoreductases distributed throughout the entire epidermis is correlated with their key role which can play in the processes of cell differentiation and cell multiplication except for those regarding keratin formation which is not produced in the epidermis of most fish. 相似文献
17.
Whole serum and elevated pH previously had been found to stimulate both cell multiplication and hyaluronic acid production by chick embryo fibroblasts in culture. In a study to determine whether cell multiplication and hyaluronic acid production both respond to a single well-defined substance, insulin was found to stimulate, and cortisol to inhibit both processes coordinately. It appears, therefore, that multiplication and differentiated function in fibroblasts respond to a common underlying regulatory signal. Inhibition of ribosomal RNA synthesis by actinomycin D does not prevent serum stimulation of hyaluronic acid production, but inhibition of total RNA synthesis does. If total RNA synthesis is inhibited only after the hyaluronic acid production has reached a new high level, it continues at that level for the next five hours. The stimulatory treatment causes an increase in the activity of the enzyme hyaluronate synthetase. Inhibition of protein synthesis prevents any increase in hyaluronic acid production, and reduces the basal level of production. Reduction of the availability of Mg2+ in the medium coordinately inhibits DNA synthesis and hyaluronic acid production. The results are discussed in the light of a model for coordinate control growth and metabolism based on the availability of Mg2+. 相似文献
18.
Gary L Bradshaw Gordon H. Sato Don B. McClure George R. Dubes 《Journal of cellular physiology》1983,114(2):215-221
A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm2, this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication. 相似文献
19.
20.
Studies on carcinogenesis by avian sarcoma viruses. VI. Differential multiplication of uninfected and of converted cells in response to insulin 总被引:24,自引:0,他引:24
H M Temin 《Journal of cellular physiology》1967,69(3):377-384
Insulin can replace the factor(s) in calf serum whose amount is limiting for multiplication in cell culture of chicken embryo fibroblasts and of chicken embryo fibroblasts infected and converted by avian sarcoma virus. In serum-free, insulin-containing medium, converted cells multiply more than do uninfected cells. It appears, therefore, that the increased multiplication in cell culture of converted cells as compared with uninfected cells results from a decreased requirement by the converted cells for an insulin-like activity found in serum. 相似文献