Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Effect of NaCI on nitrate reductase,glutamate dehydrogenase and glutamate synthase inVigna radiata calli

The effect of NaCI stress on the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) in callus lines ofVigna radiata which differ in salt resistance, was studied at weekly intervals upto 28 d of growth. After 28 d, the NaCI resistant callus (selected at 300 mM NaCI) at NaCI concentrations higher than 200 mM maintained higher NR activity than non-selected line. NaCI stress also affects aminating and deaminating activities of GDH. The NADH-GDH activity in the presence of NaCI was higher in the resistant than non-selected line. On the other hand, NAD-GDH activity in both the lines was completely inhibited after 7 d of growth. The increased activity of NADH-GDH in resistant calli may play a vital role in protecting the cells from toxic effect of increased endogenous level of ammonia which probably accumulates due to efficient NO₃ ⁻ reduction. NADH-GOGAT activity was found to decrease under salt stress in both the callus lines. Nitrogen assimilation in salt-resistant calli under salt stress was found to be characterized by high NR and NADH-GDH activities, concomitantly with low GOGAT activity. The authors are grateful to DST and CSIR for financial assistance.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.     

The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum

The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×10⁸ ml^–1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.     

The hemoglobin concentration ofChironomus cf.Plumosus l. (Diptera: Chironomidae) larvae from two lentic habitats

Hemoglobin concentrations ofChironomus cf.plumosus larvae were measured in two different habitats of the same pond. Larger larvae have higher hemoglobin concentrations than small larvae. There is strong indication that the animals of poorly oxygenated deep water, have higher hemoglobin concentrations than the animals from the well-oxygenated littoral zone.     

The Effect of Photoinhibition on Photosynthetic Oxygen Production in the Brown Alga Dictyota dichotoma

Photoinhibition of photosynthesis in the brown alga, Dictyota dichotoma, was studied with a PAM fluorometer (Walz, Effeltrich, Germany) and a homemade oxygen measuring device. As a measure of fluorescence, Fv/Fm, and for the photosynthetic yield, ΔF/Fm', were used. Oxygen measurements show clearly that the observed degree, as well as the time course, of photoinhibition depends on the fluence rate of the light used to measure changes of the production rate. After photoinhibition of photosynthesis the depression of oxygen production caused by non-saturating fluence rates was generally much more pronounced than that caused by saturating or nearly saturating fluence rates. At minimal photoinhibition the initial slope and the convexity of the fluence rate-response curve of oxygen evolution decrease, whereas the level of light saturation decreases only after strong photoinhibition. Nevertheless, at different degrees of photoinhibition, changes in the degree of the upper bending of the fluence rate-response curve of oxygen production are also linearly correlated to changes in the fluorescence ratios (Fv/Fm and ΔF/Fm'). The action spectrum of photoinhibition, calculated on the basis of changes of Fv/Fm, indicates that the reaction center of PS I is not involved in photoinhibition. The lower effectiveness of blue light in comparison to effects of green and red light may be due to chloroplast displacement, as in the so-called strong light position, the light absorbed by the thalli in vivo is decreased.     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.     

Inhibition of rabbit sperm acrosomal enzymes by gossypol

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β-N-Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol-induced sterility. © 1995 Wiley-Liss, Inc.     

Metabolic adaptation of renal carbohydrate metabolism. V.In vivo response of rat renal-tubule gluconeogenesis to different diuretics

We have studied the effects of the diuretics mersalyl, furosemide and ethacrynic acid on renal gluconeogenesis in isolated rat-kidney tubules and on the activities of the most important gluconeogenic and glycolytic enzymes in both fed and fasted rats. Mersalyl (15 mg.kg^–1 animal weight) significantly decreased the rate of gluconeogenesis in well-fed rats (68%) as well as in 24 and 48-h fasted ones (33 and 37% respectively). This inhibition occurred when lactate, pyruvate, glycerol or fructose were used as substrates. Ethacrynic acid at a dose of 50 mg.kg^–1 animal weight provoked a transient inhibition of renal glucose production by almost 20% but only in fed rats with lactate as substrate, whereas the same dose of furosemide did not affect this metabolic pathway.Parallel to these changes, mersalyl caused a significant inhibition in the maximum activity of the most important gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase, in both fed and fasted rats. Neither ethacrynic acid nor furosemide produced any variations in the activities of these enzymes. The activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase was not modified by these diuretics. Nevertheless, the activity of the thiol-enzyme glyceraldehyde 3-phosphate dehydrogenase was severely inhibited by mersalyl and to a lesser extent by the other diuretics. This inhibition was higher in fasted than fed rats. Hence, we conclude that the inhibitory effect of mersalyl on renal gluconeogenesis is due, at least partly, to a decrease in the flux through the gluconeogenic enzymes. Blood glucose was not modified after diuretic treatment in fed animals whereas mersalyl decreased the levels of blood glucose in 24-h fasted rats. Thein vivo effects of diuretics on gluconeogenesis correlate well with the previously observedin vitro effects, although ethacrynic acid was less potent as an inhibitorin vivo, probably because of its rapid clearance.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis (-aminoethylether) N,N,N,N-tetraacetic acid - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TRIS 2-amino-2-hydroxymethyl-1,3-propanediol Publication No. 166 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group, Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain