Influence of cold acclimation on membrane injury in frozen plant tissue

Cold-acclimated twigs of Amelanchier alnifolia Nutt. released less HCN at −4.5 C than nonacclimated twigs following slow freezing to −25 C or rapid freezing to −78 C. Cold-acclimated twigs frozen slowly to −25 C released more HCN than cold-acclimated twigs frozen only to −4.5 C. Cold-acclimated twigs frozen slowly to −25 C and then rapidly to −78 C released less HCN at −4.5 C than cold-acclimated twigs frozen rapidly to −78 C. In general, K⁺ efflux and the inability to reduce triphenyl tetrazolium chloride following freezing and thawing paralleled HCN release at −4.5 C. Because low K⁺ efflux and high triphenyl tetrazolium chloride reduction are known to depend upon membrane integrity, the increased K⁺ efflux and the decreased triphenyl tetrazolium chloride reduction following freezing and thawing provide indirect evidence that HCN release at −4.5 C is a measure of membrane damage in frozen cells.     

Dehydrogenase and electrochemical activity of <Emphasis Type="Italic">Escherichia coli</Emphasis> extracts

The dehydrogenase activity of Escherichia coli BB cell extracts was studied at different growth stages in the presence of different substrates and triphenyl tetrazolium chloride as an electron acceptor. It was shown that the highest degree of reduction of triphenyl tetrazolium chloride was observed during exponential growth of the bacteria when potassium isocitrate was used as a substrate. It was found that extracts of the bacteria during the exponential phase of growth on an inert glassy carbon electrode in a three-electrode liquid electrochemical cell manifested electrochemical activity in the presence of potassium citrate and methylene blue or potassium hexacyanoferrate(III) as redox mediators.     

桉树树干维管形成层和次生韧皮部热致细胞坏死的定量试验

桉树树干维管形成层和次生韧皮部热致细胞坏死的定量试验 桉树(Eucalyptus)树干暴露在森林火灾辐射热中会杀死形成层细胞及其内嵌的再生分生组织,阻止树木萌枝和恢复。目前尚无组织水平的方法来量化热处理对桉树形成层细胞活力的影响。本研究的目 标包括：(1)采用并验证四氮唑还原法检测桉树细胞活力;(2)应用该方法确定斜叶桉(Eucalyptus oblique)形成层细胞活力的阈值水平,进而确定临界温度。采用四氮唑还原法量化该桉树韧皮部-形成层细胞活力。 从斜叶桉成树上切下带有形成层和韧皮部的圆形树皮切片,将质量为1-30 mg不等的样品在20–85°C 的温 度处理中放置1分钟,并在室温条件下在0.8%的2,3,5-氯化三苯基四氮唑(TTC)中保存20–22小时以检测 细胞活力。用乙醇冷萃取得到1,3,5-三苯基四唑甲臜(TPF),在485 nm处测定吸光度。结果表明,TTC还原 法准确地量化了组织切片(包括维管形成层)中细胞活力随温度升高而下降的情况,并确定60°C 为桉树物种形成层-韧皮部细胞的临界温度。细胞活力按[TPF 处理温度]/[TPF 20°C]计算,在20-85°C之间下降90%。细胞活力结果证实,在50-70°C的温度区间,经过1分钟体外组织加热,桉树的组织坏死显著 增加。TTC 方法显示细胞活力随温度升高而下降,这与温度处理和中性红染色处理后独立获得的活细胞计 数一致。     

The use of the iron chelating agent desferrioxamine in rice cell cryopreservation: a novel approach for improving recovery

The iron chelating drug, desferrioxamine is used to suppress oxidative stress in mammalian transplant organs subjected to cold storage. The efficacy of desferrioxamine in improving post-thaw survival in cryopreserved cells from two rice culture lines was evaluated. Unfrozen rice cells maintained proliferation capacity over a fifteen day time course when exposed to concentrations of desferrioxamine > 10 mg·l⁻¹. Albeit, growth was reduced compared to controls. Short-term applications of the drug at concentrations of 0.5 and 10 mg·l⁻¹ before cryopreservation and during the early post-thaw period had a positive affect on recovery as assessed by cell proliferation and triphenyl tetrazolium chloride reduction capabilities. The pharmaceutical properties of desferrioxamine are attributed to iron sequestration and the prevention of harmful Fenton and free radical chemistry. However, desferrioxamine did not significantly reduce lipid peroxidation in cryopreserved rice cells.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

Modification of the Cellular Heat Sensitivity of Cucumber by Growth under Supplemental Ultraviolet-B Radiation

The effect of ultraviolet B (UV-B) radiation on the thermal sensitivity of cucumber (Cucumis sativus L.) was studied using UV-B-sensitive cv Poinsett 76 and UV-B-resistant cv Ashley grown under control and elevated (300 mW m-2) UV-B radiation levels. Using both cotyledon and leaf discs, the ability of the tissue to reduce triphenyl tetrazolium chloride (TTC) was determined after treatment at 50[deg]C for various times. Semilogarithmic plots of TTC reduction as a function of time at 50[deg]C were curvilinear. They were monophasic for the control cucumber and biphasic for cucumber grown in the presence of elevated UV-B. Treatment of cucumber plants at 37[deg]C for 24 h or of tissue discs at acute UV-B levels for 1 h further modified their response to elevated temperature. These results suggest that growth of cucumber under enhanced UV-B radiation levels increased its ability to withstand elevated temperatures.     

Evaluation and improvement of spectrophotometric assays of TTC reduction: maize (Zea mays) embryo as an example

The triphenyl tetrazolium chloride (TTC) reduction assay was evaluated and improved with maize seed (Zea mays cv. Zhengdan958). The reduced TTC in embryo was extracted with three kinds of organic solvents: trichloroacetic acid (TCA)/acetone, ethanol, and acetone. The absorbance spectra of the three extracts were similar, with a maximum at 485 nm. The efficiency of TCA/acetone in extracting the reduced TTC was higher than that of acetone and ethanol. A negative correlation between TTC reduction and malondialdehyde content in embryo was demonstrated. The TCA/acetone extraction may be used as a routine protocol for TTC reduction assay of seed vigor in cereal (e.g. maize, rice, wheat and barley) seeds.     

5-HT2 receptor blocker sarpogrelate prevents downregulation of antiapoptotic protein Bcl-2 and protects the heart against ischemia-reperfusion injury

Even though reperfusion is the treatment of choice in patients admitted with acute myocardial infarction, reperfusion itself has been demonstrated to activate various pathological factors especially following procedures of cardiac revascularization. 5-hydroxytryptamine (5HT) is one such factor activated during reperfusion and is known to trigger the post ischemic contractile dysfunction and pathological apoptosis. Here we demonstrate the potential effects of the 5-HT(2)A antagonist sarpogrelate in protecting the myocardium against reperfusion injury of heart. Male Wistar rats weighing between 220 and 240 g were subjected to 30 min left coronary artery (LCA) occlusion and 120 min reperfusion. Sarpogrelate (4 mg/kg) was infused intravenously for 30 min either before LCA occlusion or at reperfusion. Following reperfusion the samples were collected for infarction area, immunohistochemistry, western blotting and myocardial metabolite analysis. Sarpogrelate infusion before ischemia resulted in (a) significant recovery of post ischemic cardiac functions (LVDP, EDP), (b) significant reduction in the infarct size among the risk area after triphenyl tetrazolium chloride staining (p<0.001), (c) decreased tissue water content (p<0.05), (d) well preserved myocardial ATP (p<0.05), (e) reduction in Bcl-2 downregulation and caspase 3 activation and (g) less prevalence of apoptotic cells (3.1+/-0.4% to 15.2+/-0.6%, drug versus control). Treating the rats with sarpogrelate during reperfusion also showed similar results. This study thus demonstrates the protective effects of sarpogrelate and supports the role for 5-HT2A inhibition in preventing the reperfusion injury of the heart.     

The humification of high-moor peat

Summary It has been observed that dark coloured substances are formed by soil micro-organisms (Pseudomonas, Arthrobacter-Corynebacterium-Nocardia group, spore forming bacteria and Streptomyces) from aromatic compounds,e.g. phenol, benzoic acid, guaiacol, toluene, phthalic acid, catechol, tyrosine, and tryptophane. The dark-brown to black substances formed by these organisms possess humus-like qualities and have the appearance of dopplerite. Reducing substances reacting in the cold with triphenyl tetrazolium chloride are formed as intermediates. It is suggested that these intermediary substances are quinones, or semiquinones and that these occur as free radicals which subsequently polymerize to humus-like substances.     

Micromolar colorimetric detection of 2-hydroxy ketones with the water-soluble tetrazolium WST-1

The archetypal triphenyl tetrazolium chloride (tetrazolium red) has been used in spectrophotometric microplate assays for 2-hydroxy ketones and, by extension, for the activity of enzymes including aminotransferase, transketolase, and pyruvate decarboxylase. Better sensitivity, dynamic range, and reproducibility of this method may be achieved by (i) in situ solubilization of the colored formazan with Cremophor EL and (ii) use of the newer-generation tetrazolium salts tetrazolium violet, iodonitrotetrazolium, or WST-1 (water-soluble tetrazolium). Two to 125 μM hydroxy pyruvate, dihydroxy acetone, and indole pyruvate produced by serine:pyruvate, β-alanine (ω-amino acid):pyruvate, and aromatic amino acid aminotransferase, respectively, could be detected with WST-1.     

Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Summary Heterocysts reduce triphenyl tetrazolium chloride (TTC) faster than vegetative cells apparently because the absence of the O₂-evolving photosystem II and the high electron transport activity in these cells. Although the rate of TTC reduction in vegetative cells is increased by the continuous removal of O₂ evolved in photosynthesis, it has not been possible to obtain rates of TTC reduction comparable with those in heterocysts probably because of the continued competition for electrons between TTC and O₂. The use of nitro-blue tetrazolium chloride (NBT) as a redox indicator has revealed the presence in filaments under aerobic conditions of a gradient of electron transport activity with strongest reducing power in the heterocysts, proheterocysts and vegetative cells next to heterocysts, and with gradually diminishing activity midway between two heterocysts. This pattern is indistinct in filaments grown under micro-aerophilic conditions. The strong electron transport activity in vegetative cells adjacent to heterocysts appears to promote reducing conditions in the heterocysts. Both, red-formazan formation in the heterocysts and blue-formazan deposition in vegetative cells greatly inhibit nitrogenase activity, and this was adversely affected also by the detachment of heterocysts from vegetative cells. The findings are consistent with the idea that the association of heterocysts with vegetative cells in essential for nitrogen fixation to occur in heterocystous blue-green algae.     

Effect of Temperature, Photoperiod and Several Growth Substances on the Cold Hardiness of Chrysanthemum morifolium Rhizomes

The effect of 14 combinations of photoperiod, soil and air temperature, and growth substance applications on the cold hardiness of Chrysanthemum morifolium‘Astrid’ rhizomes was evaluated. Both triphenyl tetrazolium chloride and regrowth tests were used to determine the viability of the cold-stressed rhizome tissues. The rhizomes exhibited different degrees of cold hardiness under these environmental conditions. A combination of short photoperiod and low air and soil temperatures induced maximum cold hardiness. Low soil temperature accompanied by long photoperiods and warm aerial temperatures did not induce rhizome hardening, while some hardening in cool soils was evident under either short photoperiods or low aerial temperatures. Warm soils reduced rhizome hardening under the normally inductive short photoperiod-cool aerial conditions. Since the induction of rhizome hardening was dependent on the induction of the aerial organs, the involvement of translocatable hardiness promoters is indicated. Foliar applications of low levels of gibberllic acid (GA₃) or abscisic acid only slightly influenced rhizome hardiness.     

In Vitro Reduction of Tetrazolium Indicators by Sulfhydryl Compounds

There was considerable variation between the sulfhydryl induced in vitro reduction of the oxidation-reduction indicators (2,3,5-triphenyltetrazolium chloride, 2,3-diphenyl 5-methyl tetrazolium chloride, neotetrazolium chloride, neotetrazolium phosphate-2B, blue tetrazolium, tetrazolium violet, potassium tellurite, methylene blue, and resazurin). The neotetrazolium salts and potassium tellurite showed the greatest reducing activity. The reduction of the indicators by oxidized sulfhydryl compounds in the presence of potassium cyanide closely paralleled the reduction of the sarrte indicators by reduced sulfhydryl compounds. Iodoacetamide was the most effective sulfhydryl inhibitor as demonstrated by indicator reduction.     

Morphology and enzyme activity of nonculturable forms of Vibrio cholerae

The transition of V. cholerae into the uncultivable state under experimental conditions was accompanied by gradual changes in their morphology, motility and metabolic activity. The vibrios took the oval form, lost their flagellum, motility and enzyme activity on diagnostic media. Dehydrogenase activity tested by reduction of triphenyl tetrazolium chloride, increased at the initial stages and dropped to the initial level or even lower by the end of the observation period (10 months). Similar dynamics was noted when the cytoplasmatic marker enzyme--ATPase activity was studied. Glucose catabolism in the uncultivable forms shifted towards glycolysis. During 1-2 months ctx and tcp genes could be detected in these forms by the PCR. The dynamics of the biological properties under study made it possible to find out the existence of 3 functionally different stages in the development of an uncultivable population.     

Vital dye reaction and granule localization in periplasm of Escherichia coli

BACKGROUND: Tetrazolium salts are widely used in biology as indicators of metabolic activity - hence termed vital dyes - but their reduction site is still debated despite decades of intensive research. The prototype, 2,3,5- triphenyl tetrazolium chloride, which was first synthesized a century ago, often generates a single formazan granule at the old pole of Escherichia coli cells after reduction. So far, no explanation for their pole localization has been proposed. METHOD/PRINCIPAL FINDINGS: Here we provide evidence that the granules form in the periplasm of bacterial cells. A source of reducing power is deduced to be thiol groups destined to become disulfides, since deletion of dsbA, coding for thiol-oxidase, enhances the formation of reduced formazan. However, pervasive reduction did not result in a random distribution of formazan aggregates. In filamentous cells, large granules appear at regular intervals of about four normal cell-lengths, consistent with a diffusion-to-capture model. Computer simulations of a minimal biophysical model showed that the pole localization of granules is a spontaneous process, i.e. small granules in a normal size bacterium have lower energy at the poles. This biased their diffusion to the poles. They kept growing there and eventually became fixed. CONCLUSIONS: We observed that formazan granules formed in the periplasm after reduction of tetrazolium, which calls for re-evaluation of previous studies using cell-free systems that liberate inaccessible intracellular reductant and potentially generate artifacts. The localization of formazan granules in E. coli cells can now be understood. In living bacteria, the seeds formed at or migrated to the new pole would become visible only when that new pole already became an old pole, because of the relatively slow growth rate of granules relative to cell division.     

Variation in free-radical damage in rice cell suspensions with different embryogenic potentials

Levels of free-radical-mediated lipid peroxidation were monitored in cell-suspension cultures of Oryza sativa L. possessing different embryogenic potentials. Oxidative stress was evaluated using assays which sequentially assessed the stages of lipid peroxidation (diene conjugation, peroxidation, and the formation of secondary lipid-peroxidation products). Lipid peroxidation was significantly higher in a cell line which had lost embryogenic ability compared with lines which still retained this capacity. Superoxide dismutase (EC 1.15.1.1) activity did not vary significantly between the embryogenic and previously embryogenic lines; however, catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) activities were significantly lower in the line which had lost embryogenic ability. Metabolic activity as estimated by reduction of triphenyl tetrazolium chloride decreased with diminishing embryogenic potential and was especially low in cell lines which never exhibited embryogenic capabilities. The possible involvement of free radicals in the loss of embryogenic potential of rice cells is discussed.     

Evaluation of Viability,Shedding Pattern,and Longevity of Pollen from Genetically Modified (GM) Herbicide-tolerant and Wild-type Zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.)

Knowledge about pollen viability is important when evaluating the risk of genetically modified (GM) plants. Here, staining via iodine potassium iodide (IKI) or triphenyl tetrazolium chloride (TTC) could not distinguish between live and dead pollen from Zoysia japonica. Therefore, to obtain a reliable assessment of such viability and longevity, we developed an optimum germination medium containing 20% sucrose and 50 ppm H₃BO₃. Pollen grains transferred to the germination medium at about 1000 hours had a germination rate of >90%. Pollen was most predominantly shed at approximately 1000 hours, with viability declining to nearly 0% at 1200 hours. All germinability was lost within 150 min when stored at 25°C. No significant difference was found between GM and non-GM plants in their pollen viability or longevity.     

Facilitated enumeration of the silicate bacterium <Emphasis Type="Italic">Paenibacillus mucilaginosus</Emphasis> comb. nov. (formerly <Emphasis Type="Italic">Bacillus mucilaginosus</Emphasis>) via tetrazolium chloride incorporation into a double agar-based solid growth medium

Accurate enumeration of Paenibacillus mucilaginosus (formerly Bacillus mucilaginosus) bacterium from environmental samples on solid medium is challenging owing to its extensive extracellular polysaccharides (EPS) excretion. In the present study, P. mucilaginosus enumeration has been facilitated by a simple modification: addition of triphenyl tetrazolium chloride (TTC) to growth medium and application of a second soft agar layer. Results show distinctively better and accurate colonies’ count. This method can be applied to all bacterial species that produce excessive EPS that may interfere with direct count.     

The Azolla-Anabaena azollae relationship

The heterocystous blue-green alga, Anabaena azollae, was isolated from the leaf cavities of the water fern, Azolla caroliniana, where it occurs as an endophyte. The isolated alga was capable of light dependent CO₂ fixation and acetylene reduction. Aerobic dark acetylene reduction occurred and was dependent upon endogenous substrates. Vegetative cells of the alga reduced nitro-blue tetrazolium chloride (NBT) to blue formazan. Heterocysts did not. Heterocysts reduced triphenyl tetrazolium chloride (TTC) to red formazan faster than vegetative cells. Reduction of TTC by both heterocysts and vegetative cells was much more rapid than has been reported for free-living heterocystous blue-green algae. Both NBT and TTC inhibited acetylene reduction and CO₂ fixation. The inhibition by TTC was more closely correlated to the time of exposure of the cells to the reagent and to the amount of deposition per cell than to the number of cells containing red formazan. No differential inhibition of acetylene reduction versus CO₂ fixation was observed. Autoradiography showed that CO₂ fixation occurred only in vegetative cells. Heterocysts caused a darkening of nuclear emulsions (chemography). This observation has been employed by others as an index of reducing activity in these cells. DCMU inhibited the acetylene reducing capacity of alga isolated from dark pretreated fronds more rapidly and to a greater extent than that in alga isolated from light pretreated fronds. Ammonia in excess of 5 mM was required before any inhibition of acetylene reduction was observed under either aerobic or anaerobic conditions in the light.     

Cold tolerance in sporobolus virginicus (L.) Kunth robust form, tissue cultures and whole plants

Abstract Sporobolus virginicus (L.) Kunth robust form, is a coastal C4 grass species in tropical and subtropical regions. An artificial freeze test was used to determine the response of tissue exposed to low temperature. The response was monitored by three methods: measuring respiration rates as carbon dioxide flux before and after freezing, conducting a triphenyl tetrazolium chloride (TTC) viability assay, and observing growth of tissue cultures and the regrowth of shoots and roots from rhizome buds. The TTC assay overpredicted the survival temperature in rhizomes, when based on a 50% of control as lethal value, but was a good indicator of survival in callus and suspension cultures. Respiration rates of callus tissue declined with low temperature exposure and paralleled the TTC results. Rhizome tissue however had a more complex respiratory response. High, post-freeze, respiration rates during thawing of rhizomes at + 5.0°C were correlated with injury, detected both by a TTC assay and by the measurement of carbon dioxide flux. High respiration rates, measured after the thawing period, due to disabled rhizomes that were ultimately inviable, provide an explanation for the overprediction of the TTC assay. S. virginicus was found to be freeze-sensitive, LT₅₀= - 2.5°C, with no hardening ability and it was concluded that cellular resistance defines the limits of freezing tolerance. However, avoidance of freezing strain through an underground perenniating organ probably allows S. virginicus to survive on the polar end of its range where episodic frosts may decrease temperatures to the lower limits of the cellular tolerance of a species.