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  • Life and Medical Sciences  (32)
  • 1
    ISSN: 0749-503X
    Keywords: yeast ; Pichia stipitis ; Saccharomyces cerevisiae ; overexpression ; ARDH ; D-arabinitol dehydrogenase ; zymogram screening ; arabinitol metabolism ; xylose metabolism ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An NAD+-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a λ-cDNA library with a zymogram technique. D-Arabinitol, xylitol, D-glucitol and galactitol are substrates for the recominant protein. With D-arabinitol as substrate the reaction product is D-ribulose. The molecular weight of the native tetramer enzyme is 110 000 Da and the monomer is 30 000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85·5% identical to a D-arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D-arabinitol and P. stipitis was able to grow on D-arabinitol. The physiological role of D-rabinitol metabolism is discussed.
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  • 2
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: At 43°C (but not at 41°C), organic solvents used to dissolve water-insoluble chemotherapeutic agents become themselves lethal to cells. This finding is not unique to Chinese hamster cells (HA-1); mouse mammary sarcoma cells (EMT-6) behave similarly. The solvent concentrations involved are in the range of those needed to make drug solutions. Hence experiments measuring drug-cell interactions at elevated temperatures must include controls which independently measure solvent effects.
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  • 3
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have attempted to determine the appropriate parameter of energy status assoiciated with the survival of CHO fibroblasts under starvation conditions. Survival correlated well with adenylate energy charge (EC) but not so well with the phosphorylation potential or ATP concentration. Starved cells exhibited the capacity to resist (transiently) decreases in both EC and survival. A fall in EC was associated with decreased survival. Using this correlation, we subsequently investigated whether killing after thermal stress occurred by a mechanism analogous to starvation, perhaps due to inhibition of energy yielding pathways. This hypothesis proved to be false; over 99% of cells were killed before a decrease was observed in any of the parameters of energy status. Cells were, however, sensitized to heat under nutritionally deprived conditions, a finding which may be significant for tumor treatment by heat in vivo.
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels. © 1994 Wiley-Liss, Inc.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When cultured Chinese hamster cells were exposed to 43°C hyperthermia, effects due to glucose deprivation and to the presence of the uncoupler of oxidative phosphorylation, carbonylcyanide-3-chlorophenylhydrazone, during the 43°C treatment proved to be strongly accelerated compared to the effects at normal temperature (37°C). This strongly indicates that the availability of energy plays an important role in the response of these cells to hyperthermia. One of the reasons cells die after hyperthermia may be a lethal lack of energy. Cells heated before glucose deprivation were able to maintain viability for a longer period during deprivation than cells without the preheat treatment. As the cells might develop thermotolerance after the heat exposure, this suggests that cells in the thermotolerant state use energy in a more economical way.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 120 (1984), S. 163-168 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Prostaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PG12) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGl2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGl2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10-12 M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10-12M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a “quiescent” nongrowth state.
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Exposure of eukaryotic cells to elevated temperature leads to profound switches in cell metabolism and gene expression which may be involved in cellular homeostatic mechanisms. We have investigated the effect of heat shock (45°C) on the metabolism of the phosphoinositides, a class of phospholipids involved in the function of Ca2+-linked membrane receptors. Heat shock led to stimulation of phosphoinositide turnover in HA1-CHO and Balb C 3T3 cells, resulting in the rapid accumulation of inositol trisphosphate (lP3). Mitogenic and α1 adrenergic stimulation, with serum or phenylephrine, led to similar increases in lP3. Heat shock also caused rapid increase in phosphorylation of polyphosphoinositides (PPl). Prolonged exposure to heat 〉15 min at 45°C led to progressive cellular toxicity which was associated with depletion of PPl. This decline in PPl concentration appeared to result from inhibition of PPl resynthesis. In this respect, heat may resemble some other types of cellular stresses in stimulating membrane phospholipases to deplete classes of membrane phospholipids. The induction of PPl turnover may, therefore, be involved in both plelotropic responses to brief heat shock and toxicity resulting from prolonged thermal stress.
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  • 8
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents. © 1993 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 160 (1994), S. 249-254 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell-cell contact plays in this phenomenon. The formation of cell-cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca2+ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell-cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 128 (1986), S. 127-132 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10-6M developed after a lag of 2-3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100-fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.
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