Concanamycin 4-B: A Potent Inhibitor of Vacuolar pH Regulation in Chara Cells

Concanamycin 4-B, a macrolide antibiotic with an 18-membered lactone ring, is known as a specific inhibitor of the vacuolar type of H⁺-ATPase, as is bafilomycin A₁. The drug was tested for its effect on regulation of the vacuolar pH (pH_v) of internodal cells of a fresh water characean alga, Chara corallina, under normal conditions and under salt stress. The pH_v was measured either on isolated vacuolar sap with a conventional pH electrode or directly by inserting a pH-sensitive glass microelectrode into the vacuole. Proton-pumping into tonoplast vesicles was almost completely inhibited by concanamycin 4-B at 1 nM. Concanamycin 4-B at 1 μM significantly increased pH_v while bafilomycin A₁ was ineffective when applied at 1 μM. Concanamycin 4-B did not affect pH_v when applied at 0.1 μM and increasing the concentration to 10 μM did not amplify the degree of alkalization. Concanamycin 4-B also inhibited pH_v regulation under NaCl stress. When Chara cells were treated with 100 mM NaCl, pH_v promptly increased and then recovered to the original level. The reacidification was completely inhibited by concanamycin 4-B (1 μM), suggesting that the reacidification was achieved by the H⁺-ATPase of the tonoplast.     

Darkness improves growth and delays necrosis in a nonchlorophyllous habituated sugarbeet callus: Biochemical changes

The transfer of light-cultured green normal (N) and white habituated (HNO) sugarbeet callus to darkness reduced the growth of N callus and improved growth and delayed necrosis in the HNO callus. The decrease of dry matter of N callus under darkness was accompanied by a reduced content of carotenoids and by decreased CO₂ fixation, which was compensated by an increased dependency on externally supplied sucrose. The levels of some organic nitrogen compounds such as glutamate, proline, and free polyamines were not affected by transfer to darkness of N or HNO callus. Darkness decreased ethylene emissions in both callus types. In the HNO callus, the sucrose growth dependency and the CO₂ fixation were unaffected by darkness. Chlorophylls were absent both in light and darkness, whereas some carotenoids were accumulated in the HNO callus only in dark conditions. In another connection, a significant increase of peroxidase activity, which did not occur in the N callus, was induced by darkness in the HNO callus. A decreased content of thio-barbituric acid (TBA)-reactive substances was measured in the HNO callus transferred to darkness, whereas an increase was noticed in the N callus placed in the same conditions. These metabolic changes and the reduction of cellular damage in darkness revealed light-induced stress reactions leading to necrosis and to reduced growth of HNO callus. It appeared that darkness allowed the HNO callus to avoid the photooxidation stress. Therefore, the favorable effect of darkness on HNO growth might be explained by the suppression of photooxidative damage due to the absence of carotenoids. The higher peroxidase activity in the HNO callus maintained in darkness raised the problem of heme synthesis in this heterotrophic callus.     

Responses to recent climatewarming of Pinus sylvestris and Pinus cembra within their montane transition zone in the Swiss Alps

Abstract. Altitudinal and latitudinal distribution limits of trees are mainly controlled by temperature. Therefore climate warming is expected to induce upslope or poleward migrations. In the Swiss Central Alps, summers in the period 1982-1991 were on average 0.8 °C warmer than those of the period 30 yr before. We investigated whether populations of conifers at the montane Pinus sylvestris-Pinus cembra ecocline exhibit demographic trends in response to that warming. We found no evidence for this. Young seedlings of Pinus sylvestris, the species which is expected to expand its range upward in a warmer climate, were virtually absent from all sites, whereas large fractions of Pinus cembra populations were observed in the seedling and juvenile categories even below the present lower distribution limit of adult trees. This suggests that there are no major altitudinal shifts in response to the recent sequence of warmer summers. Germination and seedling survival trials with Pinus sylvestris suggest that temperature per se would not exclude this species even from establishing at the current treeline in the Swiss Central Alps. Similar results were found at the polar treeline. Phytotron tests of seedling survival showed much less drought resistance in Pinus sylvestris than in Pinus cembra which is in contrast to their phytogeographic distributions. Thus, the montane pine ecocline in the Swiss Central Alps seems to be stabilized by species interactions and may not be directly responsive to moderate climatic change, which needs to be taken into account in predictive attempts.     

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 M tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]_i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 M) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 M) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO₄ (400 M) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine. These results indicate that free radical-induced irreversible cell injury results from a loss of protein thiols. Changes in the cellular energy charge and lipid peroxidation do not bear a simple relationship to the survival of cardiac myocytes under oxidative stress.     

Cardiovascular, ventilatory and total activity responses of brown trout to handling stress

Changes in total activity, heart and ventilation rates were observed in 2-year-old brown trout, following handling stress, using non-contact bioelectronic monitoring equipment. Experiments were carried out in laboratory conditions at water temperatures below 4° C, Transfer between tanks as well as 5 min restraint stress increased the total activity of fish for 24 to 48 h, after which it declined to near the pre-stress level. The transfer and struggle both elevated the heart rate for 3 to 4 days. Ventilation rate was elevated to a maximum of about 30% above the nominal level and recovered within 3 to 4 days. Both heart and ventilation rates were higher in feeding fish relative to fasting fish after stress and rates remained higher throughout a 7 day period of recovery. A diel rhythm of lower rates during the night appeared in both heart and ventilation rates within 3 to 4 days after handling stress.     

Location of a gene regulating drought-induced abscisic acid production on the long arm of chromosome 5A of wheat

The accumulation of abscisic acid (ABA) by detached and partially dehydrated wheat leaves is known to be inherited in a quantitative manner. The location of genes having a major effect on drought-induced ABA accumulation in wheat was determined using a set of single chromosome substitution lines and populations derived from a cross between a high-ABA- and a low-ABA-producing genotype. Examination of a series of chromosome substitution lines of the high-ABA genotype Ciano 67 into the low-ABA recipient Chinese Spring showed that chromosome 5A carries gene(s) that have a major influence on ABA accumulation in a drought test with detached and partially dehydrated leaves (DLT). A similar DLT was used to examine ABA accumulation in a population of F₂ plants and doubled haploid (DH) lines derived from the cross between Chinese Spring (low-ABA) and SQ1 (high-ABA) in which the F₂ population (139 plants) and DH lines (96 lines) were also mapped partially with molecular markers. Analysis of variance of ABA accumulation between and within marker allele classes in the F₂ confirmed the location of a gene(s) regulating ABA accumulation on the long arm of chromosome 5A. MAPMAKERQTL showed the most likely position for the ABA quantitative trait locus (QTL) to be between the loci Xpsr575 and Xpsr426, about 8 cM from Xpsr426. A similar trend for high ABA accumulation was found in DH lines having the SQ1 allele at marker loci in the same region of chromosome 5AL, but the QTL effect was not significant. The function of the QTL is discussed.     

Manganese status,gut endogenous losses of manganese,and antioxidant enzyme activity in rats fed varying levels of manganese and fat

We hypothesized that manganese deficient animals fed high vs moderate levels of polyunsaturated fat would either manifest evidence of increased oxidative stress or would experience compensatory changes in antioxidant enzymes and/or shifts in manganese utilization that result in decreased endogenous gut manganese losses. Rats (females in Study 1, males in Study 2,n = 8/treatment) were fed diets that contained 5 or 20% corn oil by weight and either 0.01 or 1.5 μmol manganese/g diet. In study 2,⁵⁴Mn complexed to albumin was injected into the portal vein to assess gut endogenous losses of manganese. The manganese deficient rats:

Non-systemic expression of a stress-responsive maize polyubiquitin gene (Ubi-1) in transgenic rice plants

We have used the promoter, 1st exon and 1st intron of the maize polyubiquitin gene (Ubi-1) for rice transformation experiments and revealed the characteristic expression of Ubi-1 gene: (1) Ubi-1 gene is not regulated systemically but rather individual cells respond independently to the heat or physical stress; (2) Ubi-1 gene changes its tissue-specific expression in response to stress treatment; (3) the expression of Ubi-1 gene is dependent on cell cycle.     

Nucleotide sequence and expression of a ripening and water stress-related cDNA from tomato with homology to the MIP class of membrane channel proteins

The nucleotide sequence and derived amino acid sequence were determined for a full-length version of the tomato cDNA clone, pTOM75, the mRNA for which has previously been shown to accumulate in roots, ripening fruit and senescing leaves. Computer analysis of the predicted protein product, which we have named tomato ripening-associated membrane protein (TRAMP) indicates strong homology to known transmembrane channel proteins from other organisms. Northern analysis showed that this gene was induced by waterstress and that this induction was unaffected in an ABA-deficient genetic back-ground.