Warm blood cardioplegia reduces the fall in the intracellular concentration of taurine in the ischaemic/reperfused heart of patients undergoing aortic valve surgery

Summary The effect of cold and warm intermittent antegrade blood cardioplegia, on the intracellular concentration of taurine in the ischaemic/ reperfused heart of patients undergoing aortic valve surgery, was investigated. Intracellular taurine was measured in ventricular biopsies taken before institution of cardiopulmonary bypass, at the end of 30 min of ischaemic arrest and 20 min after reperfusion. There was no significant change in the intracellular concentration of taurine in ventricular biopsies taken after the period of myocardial ischaemia in the two groups of patients (from 10.1 ± 1.0 to 9.6 ±0.9mol/g wet weight for cold and from 9.3 ± 1.3 to 10.0 ± 1.3mol/g wet weight for warm cardioplegia, respectively). Upon reperfusion however, there was a fall in taurine in both groups but was only significant (P 0.05) in the group receiving cold blood cardioplegia (6.9 ± 0.8mol/g wet weight after cold blood cardioplegia versus 8.0± 0.8mol/g wet weight following warm blood cardioplegia). Like taurine, there were no significant changes in the intracellular concentration of ATP after ischaemia in the two groups of patients (from 3.2 ± 0.32 to 2.95 ± 0.43mol/g wet weight for cold and from 2.75 ± 0.17 to 2.62 ± 0.21mol/g wet weight for warm cardioplegia, respectively). However upon reperfusion there was a significant fall in ATP in both groups with the extent of the fall being less in the group receiving warm cardioplegia (1.79 ± 0.19mol/g wet weight for cold and 1.98 ± 0.27mol/g wet weight for warm cardioplegia, respectively). This work shows that reperfusion following ischaemic arrest with warm cardioplegia reduces the fall in tissue taurine seen after arrest with cold cardioplegia. Accumulation of intracellular sodium provoked by hypothermia and a fall in ATP, may be responsible for the fall in taurine by way of activating the sodium/taurine symport to efflux taurine.     

Factors influencing shoot regeneration from cotyledonary explants ofCucumis melo

Cotyledonary explants of 4-day-oldCucumis melo cv. Hale's Best Jumbo in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5 M indole-3-acetic acid and 5 M benzylaminopurine and cultured at 25–29°C under low light intensity (5–30 mol m^-2 s^-1). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.     

Photosynthesis and growth rates of Laurencia brongniartii J. Agardh (Rhodophyta, Ceramiales) in preparation for cultivation

Laurencia brongniartii is usually found at depths below 4 m, but can be found in shallow subtidal areas in crevices and on the walls of a coral reef in Amami Oshima Island, Kagoshima Prefecture, Japan, where irradiances were significantly lower than those at similar depths in open water. In preparation for the possible cultivation of this species for its antibiotic compounds, the effects of temperature and irradiance on photosynthesis and growth were measured. Photosynthesis and growth rates of L. brongniartii explants were highest at 26 and 28 °C, which closely corresponded to temperatures found during August to late December when it was most abundant. The estimated maximum photosynthesis rate (P _max) was 4.41 mol photon m^–2 s^–1 at 26 °C and 4.07 mol photon m^–2 s^–1 at 28 °C. Saturating irradiance occurred at 95 mol photon m^–2 s^–1 at 26 °C and 65 mol photon m^–2 s^–1 at 28 °C. In contrast, growth experiments at 41.7 mol photon m^–2 s^–1 caused bleaching of explants and the maximum growth rate observed during the study was 3.02 ± 0.75% day^–1 at 28 °C and 25 mol photon m^–2 s^–1. The difference in the saturating irradiance for photosynthesis and the irradiance that caused bleaching in growth experiments suggests that long-term exposure to high irradiance was detrimental and should be addressed before the initiation of large scale cultivation.     

Ecotoxicity of lead to the zebra musselDreissena Polymorpha,Pallas

The effect of lead on the filtration rate of the zebra musselDreissena polymorpha was investigated, together with the accumulation of Pb in the soft tissues of the mussels. The NOEC-filtration was 116 g.l^–1 (0,56 mol.l^–1) and the EC₅₀-filtration was 370 g.l^–1 (1.79 mol.l^–1). The NOEC-accumulation was the concentration found in the control water (1.4g.l^–1). These experiments show that the EC₅₀-filtration for Pb is similar to that for Cd, higher than that for Cu and lower than that for Zn. The water quality criteria for lead allow 25 g Pb.l^–1 in surface water. This will not cause short-term effects. Long-term effects may, however, occur, since an accumulation of Pb as low as 16 g.l^–1 was recorded in this study.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii

Fritillaria thunbergii Miq. bulb-scale sections were cultured using Murashige and Skoog (MS) medium supplemented with NAA (1.62 M) and KN/2iP/BA (0.47–23.23 M).A high frequency of bulblets was developed from the scale sections and these bulblets have developed leaves and roots in 12 weeks of culture. An optimum of 13.7 bulblets developed from scale sections on solid MS medium supplemented with 1.62 M NAA and 4.65 M KN. Cultures incubated under cycles of 16 h white fluorescent light (40 mol m^–2 s^–1) and 8 h dark at a temperature regime of 25°C have produced optimal bulblets compared to cultures incubated under continuous dark at 25°C. The bulblets were harvested at the end of culture period and were given cold treatment at 5°C for 5 weeks and then transplanted to a potting mixture of peat moss, vermiculite and perlite (1:1:1). The bulblets, which were more than 10 mm in diameter, sprouted (100%) in 5 weeks of transplantation.     

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. (Apicomplexa: Eimeriidae) from the black-capped bulbul,Pycnonotus xanthopygos

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. are described from the black-capped bulbul Pycnonotus xanthopygos from Lincoln Park Zoo, Chicago, Illinois. The bird came from southwestern Africa seven years earlier. I. ernsti oocysts are ellipsoidal to bluntly ovoid, 28–38 × 23–31m (mean 34 × 28 m) and have a single-layered oocyst wall. Micropyle, oocyst residuum and polar granules are absent. Sporocysts are elongate ovoid, 24–30 × 11–16 m (mean 27×13 m). Stieda and substiedal bodies and sporocyst residuum are present. I. blagburni oocysts are spherical to subspherical. 21–28 × 19–26 m (mean 25 × 23 m) and have a single oocyst wall. Sporocysts are ovoid and 17–23 × 10–13 m (mean 20 × 12 m). Stieda and substiedal bodies and sporocyst residuum are present.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I. Steady-state conditions

Summary The nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus quasi-linear cable theory. Under normal conditions of growth and respiration, the membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of –174 mV, the membrane conductance is 190 mhos/cm²; conductance increases to 240 mhos/cm² at –300 mV, and decreases to 130 mhos/cm² at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range.Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of –40 to –70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of 20 A/cm², a half-saturation potential near –300 mV, and a projected reversal potential of ca. –400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H⁺ extruded:1 ATP split.The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-stateI–V curves, under the assumption that cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects.The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 F/cm²) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.     

The kinetics of photoinhibition and its recovery in the red alga Porphyridium cruentum

When Porphyridium cruentum cells were illuminated with high fluence rate between 1900 and 4800 mol photons m^-2s^-1, a decrease in the photosynthetic activity of the cells was observed. Within the time frame of 20 min, and under the fluence rates studied, the sum of photons to be absorbed by cells (mg of chlorophyll (Chl), sufficient to initiate photoinhibition was calculated to be 9235.8 mol. The minimal specific light absorption rate to initiate photoinhibition in P. cruentum ranges between 2.29 and 4.26 mol photons s^-1 mg^-1 chl.a. There was a linear relationship between the specific rate of photoinhibition and the specific light absorption rate. A photon number of 2.56×10⁴ mol mg^-1 chl.a photoinhibited photosynthesis instantaneously. At 15°C, no photoinhibitory effect was observed at 2300 mol photons m^-2 s^-1 even after 45 min of illumination. At the other extreme of 35°C, 84% inhibition of photosynthetic activity was observed within 10 min of exposure to 2300 mol photons m^-2 s^-1. Between 20 and 30°C, the photoinhibitory effect was comparable. Photoinhibited P. cruentum cells recovered readily when transferred to low light (90 mol photons m^-2 s^-1) and darkness, and the specific rate of recovery was independent of the light intensity to which the cells were exposed, during the photoinhibitory treatment.Abbreviations Chlorophyll QL, specific light absorption rate Publication No. 28 of the Microalgal Biotechnology Laboratory     

Der Entwicklungscyclus mit Sexualphase bei der marinen Diatomee Coscinodiscus asteromphalus

Zusammenfassung Die Kultur der großen marinen Diatomee Coscinodiscus asteromphalus wird beschrieben. Die Synchronisation der vegetativen Stadien aus dem Entwicklungscyclus mit Sexualphase wird durch Messung der Valvendurchmesser charakterisiert. Die Art entwickelt sich von Stadien mit 200 m Valvendurchmesser (V.-D.), die nicht sexuell induzierbar sind, zu Stadien mit 80–90 m V.-D. mit einem Optimum der Induzierbarkeit und weiter zu Stadien mit 55–60 m V.-D. Bei dieser Größe ist keine weitere mitotische Zellteilung mehr möglich. Entwicklungsstadien mit 200–190 m, 140–130 m und 100–90 m. V.-D. zeigen bei 24°C und bei 18°C die gleiche Generationszeit im mitotischen Entwicklungscyclus von 1 bzw. 0,6 Zellteilungen pro Tag. Der Valvendurchmesser verringert sich bei dieser Art um 1,5 m bei 24°C und 1,4 m bei 18°C während einer Zellteilung.

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The life cycle with sexual phase in the marine diatom Coscinodiscus asteromphalus I. Culture and synchronisation of developmental stages

Summary Culture-conditions for the large marine centric diatom Coscinodiscus asteromphalus are described. The cells grow in a defined medium in a light-dark regime of 14: 10 h. Synchronization of different stages of the sexual life cycle is characterized by measuring the valve diameter (v.d.) of the cells. The cells develop from stages with 200 m v. d. (not sexually inducible) to stages with 80–90 m v. d. (optimum for sexual induction), and further to stages with 55–60 m v. d., where no following mitotic cell division is possible. The length of the pervalvar axis does not change during this development. Different stages (200–190m, 140–130 m and 100–90 m v. d.) grow with the same doubling time during their mitotic life cycle: 1 cell division per day at 24° C and 0.6 cell divisions per day at 18°C. During one cell division the valve diameter of this species decreases by about 1.5m at 24°C and by 1.4 m at 18°C. Therefore, the development from stages with 200 m v.d. to stages with 60 m v. d. takes between 90 days at 24°C and 165 days at 18°C.

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Teile einer Habilitationsschrift der Naturwissenschaftlichen Fakultät der Universität Marburg (Lahn).     

Factors affecting in vitro microrhizome production in turmeric

In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium –- MS basal medium + 0.88 M BAP (6-benzylaminopurine) + 0.92 M kinetin + 5% coconut water + 2% sucrose + 0.5% agar –- resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium –- MS basal medium + 2.2 M BAP + 0.92 M kinetin + 5% coconut water + 2% sucrose –- at 25±1°C and 16-h light (at 11.7 mol m^–2 s^–1)/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 25±1°C. Half strength MS basal medium supplemented with 80 g l^–1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2 M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1–2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1–0.4 g), medium (0.41–0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.     

TBARS,carnitine, and reduced glutathione levels in human bladder carcinoma

In this study, we investigated tissue levels of reduced glutathione (GSH) and carnitine as well as thiobarbituric acid reactive substances (TBARS, as a marker of lipid peroxidation) levels in bladder carcinoma and control group of patients. The average GSH, carnitine and TBARS levels for tumor group were respectively 7.11 ± 3.3 g/mg protein, 1.81 ± 0.39 nmol/mg protein, and 4.29 ± 3.2 mol/mg protein, versus 14.45 ± 4.11 g/mg protein, 2.14 ± 0.66 nmol/mg protein, and 2.3 ± 0.6 mol/mg protein for normal bladder tissues. Thus, tissue reduced glutathione levels (GSH) were significantly lower in patients as compared with the control group (p < 0.001) whereas average TBARS levels in the tumor group were found to be higher than those in control group. The average tissue carnitine levels in the patient group were found to be lower compared with the control group but the difference was not statistically significant (p > 0.05).     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

Mercury distribution in estuarine environments from Argentina: the detoxification and recovery of salt marshes after 15 years

Total Hg contents from abiotic (surface sediments and suspendedparticulate matter) and biological (crabs, fishes and halophytes)compartments from Bahía Blanca estuary and Mar Chiquita CoastalLagoon, Argentina, have been monitored since the 1980's. At BahíaBlanca estuary, high Hg concentrations were recorded during the early1980's in surface sediments (0.34 ± 0.22 g/g) andsuspended particulate matter (0.19 ± 0.10 g/g). Fishspecies, Mustelus schmitti (0.89 ± 0.29 g/g), Paralichthys brasiliensis (0.85 ± 0.18 g/g) and Micropogonias furnieri (0.37 ± 0.11 g/g) also presentedhigh Hg concentrations. The large industrial nucleus located within theestuary has been identified as the main metal source for this environment.Hg contents from the same area during 1996–1998 were significantlylower: surface sediments (0.164 ± 0.023 g/g), suspendedparticulate matter (0.048 ± 0.0017 g/g), fish Micropogonias furnieri (0.13 ± 0.02 g/g) and crab Chasmagnathus granulata (0.334 ± 0.071 g/g). This trendof environmental detoxification is probably related with (i) thetechnological changes incorporated by the local industry, (ii) a mostadequate management of industrial effluents, and (iii) the removal ofgreat sediment volume by dredging and refill.During the 1980's Mar Chiquita Lagoon Hg concentrations reached 0.08± 0.01 g/g in surface sediments and 0.09 ±0.025 g/g in suspended particulate matter, and 0.14 ±0.04 g/g in the fish Basilichthys bonariensis and 0.22 ±0.08 g/g in Paralichthys brasiliensis, and 0.08 ±0.01 g/g in the crab C. granulata, Hg concentrations werelower than at Bahía Blanca. Remote Hg sources for this Coastal Lagoonand atmospheric and stream transport of Hg is proposed as major Hgsources, since no Hg point sources exists nearby. Mercury concentrationsrecorded in the 1996–1998 period were lower than those recorded inthe previous decade: surface sediments (0.019 ± 0.004 g/g), suspended particulate matter (0.030 ± 0.008 g/g), halophyte Spartina densiflora (0.013 ± 0.008 g/g) or crab C. granulata (0.011 ± 0.009 g/g).Both Hg bioaccumulation and biomagnification processes were verified inBahía Blanca estuary and in Mar Chiquita Coastal Lagoon. This apparentrecovery of both estuarine environments deserves to be carefully analyzed,in order to fully understand the foundations of these processes.     

Characterization of dihydropyrimidinase from Pseudomonas stutzeri

Dihydropyrimidinase from Pseudomonas stutzeri ATCC 17588 was purified 100-fold and characterized. It was found that dihydrouracil, dihydrothymine and hydantoin could serve as substrates for the partially purified enzyme. The K _m values for dihydrouracil, dihydrothymine and hydantoin were determined to be 19.6 M, 21.3 M and 36.4 M, respectively, while their respective V _max values were 0.836 mol/min, 0.666 mol/min and 2.21 mol/min. Between pH 7.5 and 9.0, enzyme activity was shown to be maximal. The optimum temperature for enzyme activity was 45 °C. Using gel filtration, the molecular weight of the enzyme was calculated to be approximately 115000 Da. Metal ions were found to influence the level of enzyme activity. Dihydropyrimidinase activity was stimulated by magnesium ions and inhibited by either zinc or copper ions.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Doxorubicin and epirubicin iron-induced generation of free radicalsIn vitro. A comparative study

To ascertain any differences in myocardial injury exerted by the anthracyclines doxorubicin and epirubicin, their ability to generate oxygen free radicals when mixed with Fe(II) was examined in vitro using an oxygen electrode. 5–250 g/ml doxorubicin or epirubicin consumed oxygen when mixed with 50 or 100 mol/1 Fe(II). Addition of 75 mol/1 cytochrome C showed that of the consumed oxygen, approximately 80% entered the monovalent pathway of oxygen reduction. The strong inhibitory effect of 250 mg/1 catalase indicates that most of the superoxide radicals generated are further reduced to hydrogen peroxide by both anthracyclines. Addition of metal chelators DTPA (100/mol/1), or DDTC (50 mol/1) did not affect oxygen consumption, whereas EDTA (100/mol/1) or desferrioxamine (100 mol/1) with anthracyclines and Fe(II) rather stimulated oxygen consumption. It is concluded that there are no significant differences in the amount or proportion of generated oxygen free radicals between doxorubicin and epirubicin when mixed with Fe(II) in a cell-free system in vitro. Thus, the ability of the anthracyclines, in conjunction with iron alone, to generate radicals does not explain the differences of the drugs in causing myocardial injury.