The effect of ergosterol, ergocalciferol and cholecalciferol on calcium-controlled peroxidase secretion by sugarbeet cells

Provitamin D₂ (ergosterol), vitamins D₂ and D₃ reduced the calcium-mediated peroxidase secretion in three types of sugarbeet cells in suspension cultures. Vitamin D₂ was the most effective in habituated non-organogenic cells which were the less sensitive to calcium; provitamin D₂ was the most effective in habituated organogenic cells, while normal non-organogenic cells were equally sensitive to the three types of vitamin D. The calcium ionophore A23187 slightly restricted peroxidase release in all cases, except in the habituated organogenic cells in the presence of calcium where it exerted a promotive effect. The inhibiting effect of vitamin D₂ was not counteracted by the ionophore except in this habituated organogenic cell line.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

NMR natural abundance estimation of13C-metabolic solutes in normal and habituated sugarbeet cell lines

Summary NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring¹³C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of¹H,¹³C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the¹H and¹³C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these¹³C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.Abbreviations DQF-COSY double-quantum filter correlation spectroscopy - HO habituated organogenous - HNO habituated nonorganogenous - HMBC heteronuclear multiple-bond correlation - HMQC heteronuclear multiple-quantum coherence - N normal - NMR nuclear magnetic resonance - TSP sodium tetradeutero-3-(trimethylsilyl)-propionate     

Functional Correlation between Endogenous Phytohormone Levels and Hormone Autotrophy of Transformed and Habituated Soybean Cell Lines

We analysed the time course of the endogenous free IAA and cytokininlevels in hormone requiring and hormone autotrophic (both transformedand untransformed) Glycine max. L. Merr. cv. Mandarin tissuecultures. The auxin habituated line showed an enhanced endogenous IAAlevel, whereas the IAA as well as the cytokinin concentrationsin the cytokinin habituated line differed not significantlyfrom the non-habituated hormone requiring soybean callus. It were only the auxin habituated cells that could be inducedto fully habituated cells, from which a pale and a green typewas isolated. The phytohormone autotrophic growth of the paletype was sustained by enhanced IAA levels, whereas the greentype was characterised by elevated cytokinin concentrations. These results on the phytohormone content of partially and fullyhabituated soybean calli were compared with soybean crown galllines and discussed in view of the positive effect of exogenouslyapplied cytokinins on the endogenous IAA levels. ³Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw (I.W.O.N.L.) grant. ⁴Senior Research Associate Nationaal Fonds voor wetenschappelijkOnderzoek (N.F.W.O.). (Received March 25, 1988; Accepted July 7, 1988)     

Auxin-polyamine interaction with the calcium-controlled secretion of peroxidase by sugarbeet cells

Spermidine and ornithine given to normal auxin-requiring cell suspensions of sugarbeet inhibited peroxidase secretion in the absence of Ca²⁺. Habituated (organogenic or not) cells did not respond. Both compounds counteracted the Ca²⁺ - promoted enzyme secretion by three cell lines. Auxins (2,4-D and BSAA) did not modify the extracellular level of peroxidase activity in the absence of Ca²⁺ When Ca²⁺ was added, auxins increased its effect in normal cells and had practically no effect in habituated cells. The inhibitory effect of spermidine and ornithine was somewhat reduced by auxins in normal cells and increased in habituated cells. It was hypothesized that the effect of auxins did not involve the mediation of polyamines and that both types of compounds directly interacted with Ca²⁺ at the membrane level.Abbreviations BSAA [benzo(b)selenienyl-3]acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - HNO habituated non-organogenic - HO habituated organogenic - NNO normal non-organogenic     

Influence of short-chain fatty acids and osmolality on mucin release in the rat colon

Summary The influence of short-chain fatty acids (SCFA) and osmolality on mucin release in the rat colon was studied histochemically by determining number of stained mucin-containing cells. SCFA did not significantly influence the number of cells staining for mucin. Hypertonic solutions (360 mosm/l) did not affect mucin release in the proximal colon, but stimulated mucin release in the distal colon. Solutions of lower osmolality (300 or 250 mosm/l) caused a considerable release of mucin from goblet cells as well as vacuolated cells in both the proximal and the distal colon; the lower the osmolality, the more mucin was released. The mucosa of the distal colon was conspicuously affected by solutions of lower osmolality. The influence of osmolality on mucin release was entirely local.Supported by a grant from the Deutsche Forschungsgemeinschaft (En 65/9)The authors wish to thank Prof. H. Höller and G. Rechkemmer for critical advice and Miss G. Becker for technical assistanceA preliminary portion of this study was presented at the 3rd Meeting of the European Intestinal Transport Group, Southampton, 21.–23. April, 1980     

Biosynthesis and release of cell wall-like glycoproteins during the vegetative cell cycle of Chlamydomonas reinhardii

Summary The culture medium of asynchronously growing Chlamydomonas reinhardii cells contains distinct proteins which are derived from the cell walls of these cells. When cultures are synchronized by a light-dark cycle cell wall proteins are synthesized throughout the cycle, but the release of these proteins into the culture medium occurs primarily in the last quarter of the cycle, after cell separation has occurred. The mutant CW-2, which does not form a normal cell wall, continuously synthesizes and secretes cell wall proteins into the culture medium. The synthesis of cell wall protein during the cell cycle appears to be modulated and peaks of synthesis occur at the end of the light period and in the second half of the dark period, shortly after cell separation. At these times the cells devote 15% of their protein-synthetic capacity to making cell wall proteins.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid Supported by a contract from ERDA (E(04-3)-34/159) to M.J.C. and a grant from the Deutsche Forschungsgemeinschaft to W.C.L.This work was performed while W.C.L. was on leave from the University of Kaiserslautern. Dr. Lang's permanent address is Fachbereich Biologie der Universität, Pfaffenbergstraße 95, D-6750 Kaiserslautern, F. R. G. Requests for reprints may be addressed directly to W.C.L.     

Soluble and cell-wall peroxidases and auxin destruction in normal and habituated tobacco callus

Summary A comparison of the soluble and cell-wall-bound isoperoxidases of normal auxin-requiring and auxin-independent (habituated) tobacco callus revealed that normal tissues contained a higher level of isoperoxidases. There were also qualitative differences in these isoperoxidases. Partially purified soluble and ionically bound isoperoxidases of normal callus likewise exhibited higher auxin-oxidase activities. Normal tissues also were found to contain higher levels of auxin-oxidase inhibitors (auxin protectors). Overall, however, the data indicate that there is a higher rate of auxin destruction in normal tobacco callus than in habituated tissue. This presumably leads to insufficient endogenous auxin for growth. This study was supported in part by grants to T.G. from the Center IRSIA d'Etude de la Reproduction végétale and the FRFC Contract No. 2.9009. it Was carried out while T.A.T. was the holder of a senior Fellowship under the NATO senior Scientists Program.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

Summary The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.     

营养和环境因子对长春花激素自养型细胞生长和阿玛碱生成的影响

在摇瓶中用液体培养基培养长春花（Catharanthusroseus（L．）G．Don）激素自养型细胞系C20hi,比较了不同初始糖浓度、接种量、初始pH值、光照时间、光质和摇床转速对该培养细胞生长、阿玛碱积累和释放的影响。结果表明,此细胞系具有较强的环境耐受性;一定范围内初始糖浓度增加,有利于细胞生长和生物碱生成;最适的接种量是60gFW／L;一定范围内改变培养基pH值,对生物碱生成没有显著影响;照光后生物碱生成量下降,红光较蓝光更有利于生物碱生成;最适的摇床转速是120r／min。     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Caffeine eliminates gamma-ray-induced G2-phase delay in human tumor cells but not in normal cells.

It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to gamma rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the gamma-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca(++) from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca(++)-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.     

Effects of sodium butyrate on proliferation-dependent insulin gene expression and insulin release in glucose-sensitive RIN-5AH cells.

A rat islet tumor subclone, RIN-5AH-T2-B, was cultured with 2 mmol/liter of the proliferation-arresting compound sodium butyrate (NaB). Insulin gene expression and glucose-stimulated insulin release were analyzed and compared with logarithmically proliferating and confluent control cells cultured without NaB. Logarithmically proliferating control cells revealed high insulin gene expression. In the presence of amino acids, these cells showed a dose-dependent insulin response to glucose with a half-maximal and maximal 6.5-fold stimulation by 0.8 and 5.6 mmol/liter D-glucose, respectively. However, as the control cells approached growth arrest, insulin gene expression subsided to below detectability, an occurrence that is associated with decreased insulin release and accumulation of cells in the G1 phase of the cell cycle. In contrast, NaB-arrested cells showed continuous insulin gene expression throughout the experiment. Despite this, insulin release in response to glucose was lost. NaB revealed a biphasic effect on the cell-cycle: after an initial leaky G1 arrest during the first 24 h, the 5AH-B cells were arrested in G2 during the following 3 days. These data suggest that insulin gene expression and glucose-stimulated insulin release are affected by the cell cycle. These glucose-sensitive RIN-5AH-T2-B cells may be useful in studies of insulin secretion and gene regulation.     

Comparative study of respiratory metabolism in three types of sugarbeet calli,a normal and two habituated ones (organogenic and non-organogenic)

The respiratory metabolism was studied in three types of sugarbeet (Beta vulgaris L. var. altissima) calli: a normal callus (N) and two fully habituated (auxin- and cytokinin-independent) calli, organogenic (HO) and non-organogenic (HNO). Except for the HO callus at day 14, the oxygen consumption rates of the habituated calli were always higher than that of the normal callus throughout the cycle of culture. The maximum activity of the cyanide-resistant pathway (alternative pathway) was much higher in the two habituated calli than in the normal one. By contrast, important differences were found in HNO and HO calli concerning the activity of the cytochrome pathway. In HNO cells, the high activity of this pathway was correlated with a high ATP level while the inverse situation was observed in HO cells. The physiological significance of these results is discussed.     

Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated.Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C).The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred.The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.     

Polyamine levels in relation to growth and NaCl concentration in normal and habituated sugarbeet callus cultures

Abstract. The concentrations of putrescine, spermidine and spermine, the only polyamines detectable in normal and habituated calli of Beta vulgaris L. ssp. altissima , were much higher in the habituated callus than the normal callus, irrespective of experimental conditions. These results suggest that, in normal (tolerant to NaCl) and habituated (sensitive to NaCl) calli, there exists a competition for the common precursor of ethylene and polyamine biosynthesis viz. S-adenosylmethionine. A disequilibrium favouring the synthesis of putrescine and spermidine in the habituated callus might be linked to structural deterioration of the cell membrane following extended culture or severe osmotic stress (68 mol m⁻¹ NaCl). The maintenance of membrane integrity by the normal callus coincides with ethylene production at the expense of polyamine synthesis. In contrast to the habituated callus, the salinity tolerance of the normal callus is accompanied by the accumulation of proline under hypersaline conditions (274mol m⁻³). The important osmoregulatory role played by quaternary ammonium compounds in the-aerial parts of Chenopodiaceae, especially the sugarbeet, is not observed in the calli, these compounds being found in very low concentrations in saline conditions.     

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.     

Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituated Escherichia coli

A study of the conjugal transfer of ColV,I-K94 tn10 from acid-treated donors suggested that acid-habituated recipients repair acid-damaged plasmid DNA better than those that are not habituated. The presence of an increased repair activity for acid-damaged DNA in habituated cells was confirmed by isolating pBR322 from acid-treated organisms; habituated cells produced more transformants when transformed by it than did non-habituated ones. Additionally, agarose gel electrophoretic studies of pBR322 DNA isolated from acid-damaged cells and tests of its transforming activity both indicated that plasmid DNA in habituated cells is less damaged by extreme acidity than is that in non-habituated organisms.