Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.     

Effect of Furostanol Glycosides from Dioscorea deltoidea on Redox State of Alfalfa Cells In Vitro

Russian Journal of Plant Physiology - The effect of exogenous furostanol glycosides (FG) on the activity of redox enzymes was investigated in suspension cell culture of alfalfa (Medicago sativa...     

Estradiol Stimulates Vasodilatory and Metabolic Pathways in Cultured Human Endothelial Cells

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.     

Ion Composition of Tobacco Cells Cultured under Sulfur Deficiency

In both photoheterotrophic and heterotrophic tobacco cells areduced supply of sulfate in the medium did not alter the ratebut the duration of exponential growth. The higher the sulfatesupply in the medium the longer exponential growth proceeded.However, the ion composition of photoheterotrophic and heterotrophiccells was affected by sulfur deficiency in completely differentways. The dynamics in the K⁺-, Na⁺-, Mg²*-, nitrate-, phosphate-,and malate-con-tents of photoheterotrophic cells during growthwere not at all, or only slightly changed, when the sulfatesupply in the medium was reduced from 1.8mM to 1.2 mM, 0.6 mM,or 0.3mM. In heterotrophic tobacco suspensions, however, severesulfur deficiency caused K⁺, Na⁺, Mg²⁺, and malate to accumulateand nitrate to begin to accumulate earlier inside the cells.Addition of sulfate after 4 days to heterotrophic suspensionsgrown under sulfur-limiting conditions prevented the accumulationof these cations and anions. During the initial period of growthalso phosphate accumulated inside heterotrophic tobacco cellsto amounts found to be the higher the smaller the sulfate-contentof the media. Apparently, in photoheterotrophic tobacco cellsthe ion composition can homeostatically be regulated independentfrom the cells' sulfate supply, whereas the ion compositionof heterotrophic tobacco cells appears to be highly dependenton the sulfate supply of the cells. ⁴Present address: Fraunhofer Institut für AtmosphärischeUmwéltforschung, Kreuzeckbahnstr. 19, D-8100 Garmisch-Partenkirchen, F.R.G. (Received August 30, 1988; Accepted January 18, 1989)     

Amyloid β Protein Primes Cultured Rat Microglial Cells for an Enhanced Phorbol 12-Myristate 13-Acetate-Induced Respiratory Burst Activity

Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide-induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in Aβ(1–40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.     

X-Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra-structural changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.     

Effects of Lipids on ENaC Activity in Cultured Mouse Cortical Collecting Duct Cells

Direct effects on epithelial Na⁺ channels (ENaC) activity by lipids, e.g., arachidonic acid (AA), eicosatetraynoic acid (ETYA), linoleic acid (LA), stearic acid (SA), hydroxyeicosatetraenoic acid (HETE), 11,12–epoxyeicosatrienoic acid (EET), (PGF2), and (PGE2), in cultured mouse cortical collecting duct (M1) cells were clarified by using single-channel recordings in this study. In a cell-attached recording, a bath application of 10 μM AA significantly reduced the ENaC open probability (NPo), whereas 10 μM ETYA or 5 μM LA only induced a slight inhibition. The inside-out recording as a standard protocol was thereafter performed to examine effects of these lipids on ENaC activity. Within 10 min after the formation of the inside-out configuration, the NPo of ENaC in cultured mouse cortical collecting duct (M1) cells remained relatively constant. Application of ETYA or LA or SA exhibited a similar inhibition on the channel NPo when applied to the extracellular side, suggesting that fatty acids could exert a nonspecific inhibition on ENaC activity. 11,12-EET, a metabolite of AA via the cytochrome P450 epoxygenase pathway, significantly inhibited the ENaC NPo, whereas 20-HETE, a metabolite of AA via the hydroxylase pathway, only caused a small inhibition of the ENaC NPo, to a similar degree as that seen with ETYA and LA. However, both PGE2 and PGF2α significantly enhanced the ENaC NPo. These results suggest that fatty acids exert a nonspecific effect on ENaC activity due to the interaction between the channel proximity and the lipid. The opposite effects of 11,12-EET and prostaglandin (PG) implicate different mechanisms in regulation of ENaC activity by activation of epoxygenase and cyclooxygenase.     

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA^r) was selected from 6 × 10⁸ suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA^r cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA^r cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.     

Photosynthetic Characteristics and Carbonic Anhydrase Activity in Cells Cultured Photoautotrophically and Mixotrophically and Cells Isolated from Leaves

Cultured green cells of Nicotiana tabacum var. Samsun, Cytisusscoparius Link and Hyoscyamus niger which were grown photoautotrophicallyunder a stream of air enriched with 1% CO₂ or mixotrophicallyin the presence of 3% sucrose and ordinary air showed very lowcarbonic anhydrase activity, which was only 0–9% of thatin the respective intact leaves. The CO₂ compensation pointfor photosynthesis of autotrophically and mixotrophically culturedgreen cells of tobacco was higher than 0.3 mM NaHCO₃ at pH 7.8,but that of the cells isolated from tobacco leaves was lowerthan 0.1 mM NaHCO₃ at pH 7.8. The fact that the cultured cellscannot grow photoautotrophically under ordinary air is due toa high CO₂ compensation point in photosynthesis. The dark respiratoryactivity in both photoautotrophically and mixotrophically culturedtobacco cells was more than 7-fold that in the cells isolatedfrom tobacco leaves. We therefore could not conclude whetherthe high CO₂ compensation point in the cultured cells is dueto the low carbonic anhydrase activity or simply reflects thehigh respiratory activity. (Received July 10, 1980; Accepted November 25, 1980)     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

Effect of Inorganic Phosphate on the Extracellular Acid Phosphatase Activity of Tobacco Cells Cultured in vitro

Acid phosphatase activity in culture medium of tobacco cells growing in suspension increased with the age of the culture from which the medium was obtained. The increase in the activity was accelerated by omitting inorganic phosphate from nutrient medium, and it was depressed by addition of inorganic phosphate or cycloheximide. Amylase and β-galactosidase activities were not induced by the omission of inorganic phosphate. It was concluded that derepression of acid phosphatase synthesis was involved in the increase in the extracellular acid phosphatase activity upon inorganic phosphate depletion.     

The Respiratory Activity of Rhodococcus rhodochrousM8 Cells Producing Nitrile-Hydrolyzing Enzymes

The respiratory activity of Rhodococcus rhodochrousM8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. The culturing of cells with acrylonitrile and acrylamide yielding maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01–0.5 mM acrylonitrile, 0.025–1.0 mM acetonitrile, and 0.01–0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

The Effect of pH on Glycolysis and Phosphofructokinase Activity in Cultured Cells and Synaptosomes

Abstract: Several G_i-linked neurotransmitter receptors, including dopamine D₂ receptors, act synergistically with Ca²⁺-mobilizing stimuli to potentiate release of arachidonic acid (AA) from membrane phospholipids. In brain, AA and its metabolites are thought to act as intracellular second messengers, suggesting that receptor-dependent potentiation of AA release may participate in neuronal transmembrane signaling. To study the molecular mechanisms underlying this modulatory response, we have now used Chinese hamster ovary cells transfected with rat D₂-receptor cDNA, CHO(D₂). Two antisense oligodeoxynucleotides corresponding to distinct cDNA sequences of cytosolic, AA-specific phospholipase A₂ (cPLA₂) were synthesized and added to cultures of CHO(D₂) cells. Incubation with antisense oligodeoxynucleotides inhibited D₂ receptor-dependent release of AA but had no effect on D₂-receptor binding or D₂ inhibition of cyclic AMP accumulation. In addition, pharmacological experiments showed that D₂ receptor-dependent AA release was prevented by nonselective phospholipase inhibitors (such as mepacrine) but not by inhibitors of membrane-bound, non-AA-specific PLA₂ (such as p -bromophenacyl bromide). cPLA₂ is expressed in brain tissue. The results, showing that cPLA₂ participates in receptor-dependent potentiation of AA release in CHO(D₂) cells, suggest that this phospholipase may serve a similar signaling function in brain.     

Static Magnetic Field Influence on the Activity of some Respiratory Enzymes in Wheat

Most of the published studies on magnetic field action on biological systems have examined reactions in animals, while a smaller number of studies have reported magnetic field effects in plants. The effects of static magnetic field on the activity of several key enzymes in plant metabolism, such as malate dehydrogenase, succinate oxidase, succinate dehydrogenase, and cytochrome oxidase in young wheat seedlings, have been investigated in this study. It appears that the observed changes in enzyme activity could be considered to be a result of the influence of the magnetic field on the reactivity of these enzymes, including effects on metal cations that regulate enzyme activity. The results support the idea of the existence of “biological windows,” particularly with respect to exposure time.     

Xanthine Oxidase/Dehydrogenase Activity in Intact Cultured Cells (In Situ Analysis)

The measured ratio of xanthine oxidase activity to the total activity of xanthine oxidase and dehydrogenase showed higher values in intact cells than when similar cells were homogenized. The total activity was the same for both systems. The xanthine oxidase ratio was 90, 60, 50, 50, 60% in V79, RIF/Ha³, SCC7, KHT intact cells and freshly extracted murine peritoneal macrophages respectively while the corresponding ratios measured were 25, 40, 38, 35, 22% when the cells were lysed by homogenization. Superoxide radical 0₂ production by addition of xanthine to intact or homogenized cells to activate intracellular xanthine oxidase was higher in intact than homogenized cells. Homogenization of cells and tissues in the presence of dithioerythritol (DTE) can evidently lead to a considerable under-estimation of the xanthine oxidase ratio. The effect of hypoxia on cells has also been examined.     

胡桐悬浮培养细胞中苯丙氨酸解氨酶活性与红厚壳素产量的关系

胡桐CR2细胞悬浮培养过程中,苯丙氨酸解氨酶(PAL)活性上升后红厚壳素产量即增加.加入真菌诱导子后,PAL活性与红厚壳素产量呈一定的正相关.以PAL抑制物降低PAL活性后,红厚壳素产量也降低;诱导物与抑制物同时加入,PAL活性与红厚壳素产量均界于诱导物处理与未处理之间.