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941.
D. Needham 《Cell biochemistry and biophysics》1991,18(2):99-121
Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior
of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link
between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout
the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated
with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed
throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent
cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm3 to 5700 μm3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation
of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic
cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates
that a given cell line cannot be characterized by a single value for any one property, and that properties must be related
to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence
of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For
example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear
conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features
in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments
directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic
forces of the circulatory system. 相似文献
942.
Hiroyoshi Hoshi Yuji Takagi Keizo Kobayashi Masakazu Onodera Taneaki Oikawa 《In vitro cellular & developmental biology. Animal》1991,27(7):578-584
Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated
culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2),
lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent
cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of
fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or
BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed
in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After
granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling
level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations
(14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation
of bovine granulosa cells. 相似文献
943.
Javier Turnay Nieves Olmo José G. Gavilanes María A. Lizarbe 《In vitro cellular & developmental biology. Animal》1991,27(6):447-452
Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell
culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology;
b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened
morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived
fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells.
Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III
collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as
a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent
phenotypic alteration in the tumor-associated fibroblastlike cells. 相似文献
944.
Summary A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the
culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE)
emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics
of normal epithelial cells. The cells formed a small cyst consisting of keratined squamous epithelium in syngenic hosts. A
screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that
epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity
was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell
line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent
of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation,
and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8
and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results
constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to
malignant cells, and suggest that conversion from a state of cell cycle-dependent autocrine expression of one or more members
of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy.
The work was supported in part by grants CA37589 and DK35310 to Dr. McKeehan, from the National Cancer Institute, Bethesda,
MD. 相似文献
945.
Maryceline T. Espanol Lawrence Litt Guo-yuan Yang Lee-Hong Chang Pak H. Chan Thomas L. James Philip R. Weinstein 《Journal of neurochemistry》1992,59(5):1820-1828
Metabolic tolerance of low intracellular pH (pH(i)) was studied in well-oxygenated, perfused, neonatal, rat cerebrocortical brain slices (350 microns thick) by inducing severe hypercapnia. In each of 17 separate experiments 80 brain slices (approximately 3.2 g wet weight) were suspended in an NMR tube, perfused with artificial CSF (ACSF), and studied at 4.7 T with 31P and 1H NMR spectroscopy. Spectra obtained every 5 min monitored relative concentrations of lactate or high-energy phosphate metabolites, from which pH(i) and extracellular pH were determined. Unperturbed slice preparations were metabolically stable for > 10 h, with no significant changes occurring in pHi, ATP, phosphocreatine (PCr), inorganic phosphate, or lactate. Different levels of hypercapnia were produced by sequentially perfusing slices with the following different ACSF batches, each having previously been equilibrated with a specific mixture of CO2 in oxygen: (a) 10% CO2, 15 min of perfusion; (b) 30% CO2, 15 min of perfusion; (c) 50% CO2, 15 min of perfusion; (d) 70% CO2, 30 min of perfusion; (e) 50% CO2, 15 min of perfusion; (f) 30% CO2, 15 min of perfusion; and (g) 10% CO2, 15 min of perfusion. At the completion of this protocol slices were again perfused with fresh ACSF that was equilibrated with a 95% O2/5% CO2 gas mixture. In each of five separate 1H and 31P experiments, brain slices were recovered within 2 h after termination of exposure to high CO2. The pHi was determined from measurements of the chemical shift difference between phosphoethanolamine and PCr, using a calibration curve obtained for our preparation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
946.
Specific Expression of N-Acetylaspartate in Neurons, Oligodendrocyte-Type-2 Astrocyte Progenitors, and Immature Oligodendrocytes In Vitro 总被引:14,自引:0,他引:14
Jutta Urenjak Stephen R. Williams David G. Gadian Mark Noble 《Journal of neurochemistry》1992,59(1):55-61
To test the specificity of N-acetylaspartate (NAA) as a neuronal marker for proton nuclear magnetic resonance (1H NMR) spectroscopy, purified and characterized cultured cells were analyzed for their NAA content using both 1H NMR and HPLC. Cell types studied included cerebellar granule neurons, type-1 astrocytes, meningeal cells, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, and oligodendrocytes. A high concentration of NAA was found in extracts of cerebellar granule neurons (approximately 12 nmol/mg of protein), whereas NAA remained undetectable in purified type-1 astrocytes, meningeal cells, and mature oligodendrocytes. However, twice the neuronal level of NAA was found in O-2A progenitors grown in vitro. In addition significant levels of NAA were also detected in cultures of immature oligodendrocytes. Our data partly support previous suggestions that NAA may be a useful neuronal marker for 1H NMR spectroscopic examination of the adult brain. However, they also raise the further possibility that alterations of NAA associated with some specific brain disorders, particularly disorders seen in newborn and young children, may reflect abnormalities in the development of oligodendroglia or their precursors. 相似文献
947.
A G(o) type G protein distinct from the major species of G(o) was recently isolated from bovine brain and designated G(o)*. The cDNAs encoding two forms of mammalian G(o) alpha were also isolated and designated GoA alpha and GoB alpha. To recognize two forms of G(o) type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoA alpha (SNTYEDAAAYIQTQF) and GoB alpha (TEAVAHIQGQYWSK). Purified anti-GoA alpha antibodies reacted with the major species of G(o) alpha purified from bovine and rat brain, whereas anti-GoB alpha antibodies reacted only with rat G(o)*alpha, but not with the major species of G(o) alpha or bovine G(o)*alpha. These results indicate that the major species of G(o) alpha is encoded by GoA alpha cDNA and G(o)*alpha is encoded by GoB alpha cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoA alpha seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoB alpha were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC12, NG108-15, C6, GA-1, G8, and 3T3-L1 cells. Treatment of PC12 cells with nerve growth factor caused the extension of neuron-like processes and the increase in the level of GoA, but not in the level of GoB. 相似文献
948.
Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids. 相似文献
949.
Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors. 相似文献
950.
A. Fatatis A. Bassi M. R. Monsurrò G. Sorrentino G. D. Mita G. F. Di Renzo L. Annunziato 《Journal of neurochemistry》1992,59(1):1-9
The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin. 相似文献