Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells

Abstract: The effect of heat shock on agonist-stimulated intracellular Ca²⁺ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca²⁺], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca²⁺]_i rise was abolished and the [Ca²⁺]_i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca²⁺-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP₃) production was not altered by heat shock at 12 h after heat shock, whereas IP₃ receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP₃ receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs-mediated intracellular Ca²⁺ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca²⁺]_i rise.     

The effect of root temperature on the induction of nitrate reductase activities and nitrogen uptake rates in arctic plant species

We investigated whether six arctic plant species have the potential to induce nitrate reductase (NR) activity when exposed to NO₃ ^--nitrogen under controlled environment conditions, using an in vivo assay that uses the rate of NO₂ ^--accumulation to estimate potential NR activity. We also assessed the effect of low root temperatures on NR activity, growth and nitrogen uptake (using ¹⁵N applications) in two of the selected species. Five of the six species (Cerastium alpinum, Dryas intergrifolia, Oxyria digyna, Saxifraga cernua and Salix arctica) were capable of inducing NR activity when exposed to solutions containing 0.5 mM NO₃ ^- at 20°C for 10 days. Although in vivo NR activity was not induced in Saxifraga oppositifolia under controlled conditions, we conclude that it was capable of growing successfully on NO₃ ^-, due to the presence of moderate rates of NR activity observed in both NH₄ ⁺-grown and NO₃ ^--treated plants. Exposure of O. digyna and D. integrifolia to 3°C root temperatures for two weeks, with the shoots kept at 20°C, resulted in root and leaf NR activity rates of NO₃ ^--treated plants being reduced to rates exhibited by NH₄ ⁺-grown plants. Although these decreases in NR in both species appeared to be due to limitations in NO₃ ^--uptake and growth rate (rather than direct low-temperature inhibition of NR synthesis per se), direct low-temperature inhibition of root NR synthesis could not be ruled out. In contrast to the temperature insensitivity of NH₄ ⁺ uptake in D. integrifolia, NO₃ ^--uptake in D. integrifolia was inhibited by low root temperatures. We conclude that the selected arctic species have the genetic potential to utilize NO₃ ^--nitrogen, and that low root temperatures, in conjunction with other environmental limitations, may be responsible for the lack of induction of NR in D. integrifolia and Salix arctica under field conditions.     

The fate of spring applied fertilizer N during the autumn-winter period: comparison between winter-fallow and green manure cropped soil

A field experiment was carried out at a pilot plot that was cropped with oilseed rape, and then left partly fallow and partly cropped with a green manure (mustard) during the autumn after harvest of the oilseed rape. The rape residues were incorporated in the soil. Methods used to quantify the N fluxes from harvest until sowing of the next crop were (1) ¹⁵N balance method, (2) total mineral N analysis and (3) NO emission measurements. Losses of spring applied fertilizer N were negligible in cropped plots and minimal in fallow plots during the following autumn-winter period. Most of the plant-N residues was retained by the organic N pool of the upper 30-cm soil layer. The green manure contributed slightly to soil available N at sowing of the next crop. However, the incorporation of plant material resulted in a nitrate flux that was at risk of leaching on the fallow plots, and on the green manure plots after incorporation of the green manure. This nitrate was largely derived from soil organic N, not from unused fertilizer applied in spring or from immobilized fertilizer. The NO emissions from the green manure plots were significantly higher than emissions from the fallow plots. The plants had a stimulating effect on the NO emission. A relationship between the NO emission and the soil nitrate concentration could not be established. No emissions were measured after green manure incorporation due to the low temperatures at the pilot plot. However, a greenhouse experiment showed an increased emission after incorporation. The NO emissions seemed to be related with the soil ammonium concentration.     

Biological nitrogen fixation in four Gliricidia sepium genotypes

A field experiment was conducted at the Coconut Research Institute in Sri Lanka to examine the biological nitrogen fixation potential of three Gliricidia sepium provenances (OFI 14/84, 17/84, 12/86) and a local landrace (designated LL), using the ¹⁵N isotope dilution method. There was marked variation in dry matter, total N, nodulation and ¹⁵N enrichment among the Gliricidia genotypes (=0.001), and the dry matter yield of Cassia siamea (syn. Senna siamea), the non-N₂ fixing reference plant was higher than for G. sepium. In all cases, highest biomass and total N were aboveground, with roots on average accounting for < 20 % of total dry matter or the total N in plants. Atom % ¹⁵N excess was highest in C. siamea, and lowest in OFI 14/84. Although atom % ¹⁵N excess was lower in Gliricidia leaves than in the other organs (all of which had similar ¹⁵N enrichments), values of % N derived from atmospheric N₂ fixation (% Ndfa) calculated for any individual organ or for the whole plant were similar. This was because the relative distribution of ¹⁵N in the different parts of the fixing plant followed the same trend as in the reference plant. There were significant differences (p=0.01) in N₂ fixation between the Gliricidia genotypes. The values ranged from 17.8 g N tree^-1 (equivalent to 86 kg N ha^-1 at 5000 trees ha^-1) in OFI 12/86 to 61.7g N tree^-1 (equivalent to 309 kg N ha^-1) in OFI 14/84. Although most of this variability was due to differences in both % Ndfa and total N in plant, amount of N fixed was more correlated with total N in plant (r=0.935) than with % Ndfa (r=0.707). On average, % Ndfa in all three G. sepium provenances and LL was about 55 % or 34.6 g N tree^-1 (equivalent to some 166 kg N ha^-1) in the 9 months within which N₂ fixation was measured. This represents a substantial contribution of N into the soil-plant system.     

Carbon distribution and nitrogen partitioning in a soil-plant system with barley (Hordeum vulgare L.), ryegrass (Lolium perenne) and rape (Brassica napus L.) grown in a14CO2-atmosphere

To examine the influence of plant-microorganism interactions on soil-N transformations (e.g. net mineralization, net immobilization) a pot experiment was conducted in a¹⁴C-labelled atmosphere by using different (two annuals, one perennial) plants species. It was assumed that variation in below-ground, microorganism-available C would influence N transformations in soil. Plant species were fertilized (low rate) with¹⁵N-labelled nitrogen and grown, during days 13 and 62 after germination, in a growth chamber with a¹⁴C-labelled atmosphere. Nitrification was inhibited by using nitrapyrin (N-Serve). During the chamber period, shoots were harvested, and associated roots and soil were collected on two sampling occasionm, e.g. after 4 and 7 weeks in the growth chamber.The distribution of net (%) assimilated¹⁴C was significantly affected by both plant and time factors, and there was a significant plant × time interaction. There were significant differences between plants in all plant-soil compartments examined as well as in the degree of the plant × time interaction.Differences in the¹⁴C distribution between plants were due to both interspecific and developmental variation. In general, when comparing¹⁵N and¹⁴C quantities between species, many of the differences found between plants can be explained by the differences determined in the weight of shoot or root parts. Despite the fact that amounts of C released were greater in ryegrass than in the other plant-treatments no unequivocal evidence was found to show that the effects of plant-microorganism interactions on soil-N mineralization were greater under ryegrass. Possible mechanisms accounting for the partitioning of N found among plant biomass, soil biomass and soil residues are discussed.     

Inhibition of bioenergetics alters intracellular calcium,membrane composition,and fluidity in a neuronal cell line

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca²⁺-pump activity measured by⁴⁵Ca²⁺ uptake was >92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (³H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca²⁺ from the incubation medium ablated the CN/2-DG induced release of³H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (_c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca²⁺-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca²⁺ as a result of Ca²⁺-pump inhibition and thereby activate Ca²⁺-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.     

^15N标记稻草中N,C在羊体内的转化和利用

应用~(13)N标记稻草饲喂3只山羊,以探明羊对稻草N、C化合物的消化、吸收、排泄和转化规律。结果表明,已宰杀的2只羊消化、吸收、转化为羊机体的~(15)N占试验日粮中~(15)N富集总量的38.54和23.78％,平均为31.16％。3只羊从粪尿中排泄的~(15)N各占饲料中~(15)N的34.78、33.88和33.18％,平均为33.95±0.80％,已屠宰的2只羊对饲料~(15)N总回收率为73.32和56.96％,损失率为26.68和43.04％。饲料~(15)N的回收利用率与饲料中氨基酸的消化率(％)相吻合。1、2、3号羊对饲料碳水化合物的消化率分别为76.40、68.66和65.19％。其中饲喂2、3号羊的饲料中都含稻草50％左右,羊对碳水化合物的平均消化率为66.93％。     

Syntaxin-5's flexibility in SNARE pairing supports Golgi functions

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE-mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v-SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG-deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass-spectrometry analysis of STX5-interacting proteins revealed two novel Golgi SNARE complexes—STX5/SNAP29/VAMP7 and STX5/VTI1B/STX8/YKT6. These complexes are present in wild-type cells, but their usage is significantly increased in both GS28- and COG-deficient cells. Upon GS28 deletion, SNAP29 increased its Golgi residency in a STX5-dependent manner. While STX5 depletion and Retro2-induced diversion from the Golgi severely affect protein glycosylation, GS28/SNAP29 and GS28/VTI1B double knockouts alter glycosylation similarly to GS28 KO, indicating that a single STX5-based SNARE complex is sufficient to support Golgi glycosylation. Importantly, co-depletion of three Golgi SNARE complexes in GS28/SNAP29/VTI1B TKO cells resulted in severe glycosylation defects and a reduced capacity for glycosylation enzyme retention at the Golgi. This study demonstrates the remarkable plasticity in SXT5-mediated membrane trafficking, uncovering a novel adaptive response to the failure of canonical intra-Golgi vesicle tethering/fusion machinery.     

Enantiomeric LC separation of calcium antagonists on protein-based chiral stationary phases

Three chiral calcium antagonist drugs, bepridil and two dihydropyridine derivatives (nicardipine and REC 15/2375), have been successfully separated within short retention times using either the α₁-acid glycoprotein chiral stationary phase (Chiral AGP) or the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component (propan-2-ol, acetonitrile, ethanol) in order to modulate the retention properties of each system. The influence of pH and percentage of organic modifier on retention, selectivity, resolution, and column performance are discussed for bepridil analyzed on Chiral AGP and for the two dihydropyridines (nicardipine and REC 15/2375) analyzed on Ultron ES-OVM stationary phases. © 1993 Wiley-Liss, Inc.     

A comparison of the effects of subsoiling on plant uptake and leaching losses of sulphur and nitrogen from a simulated urine patch

Synthetic cow urine labelled with ³⁵S and ¹⁵N was applied to large, undisturbed, monolith lysimeters sampled from subsoiled and non-subsoiled areas of a grass/clover pasture. For one year following the urine application, the lysimeters were subjected to a combination of natural rainfall, simulated rainfall and simulated flood irrigations. Drainage from the lysimeters was sampled regularly and monthly (approx.) pasture cuts taken. At the end of the year, the lysimeters were destructively sampled in 50 mm depth increments for soil analysis. Leachates, plant samples and soil samples were analysed for ³⁵S and ¹⁵N.There were no significant differences in plant uptake of ³⁵S and ¹⁵N between the subsoiled and nonsubsoiled lysimeters. Initially grass showed a higher degree of labelling than clover. Total amounts of ³⁵S and ¹⁵N leached from the subsoiled lysimeters were approximately twice that leached from the nonsubsoiled ones. Leaching patterns differed substantially between the two nutrients.Total recoveries of ³⁵S (in plants, leachates and soil extracts) accounted for 82% of the applied ³⁵S for the subsoiled lysimeters and 72% for non-subsoiled ones. The unrecovered ³⁵S is considered to have been incorporated into soil organic matter. Total recoveries of ¹⁵N (in plants, soil and leachates) were similar to those for ³⁵S, but unrecovered ¹⁵N is attributed to loss by denitrification.