The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Antioxidant Activity of 5-Hydroxytryptophan, 5-Hydroxyindole, and Dopa Against Microsomal Lipid Peroxidation and Its Dependence on Vitamin E

The antioxidant capacity of 5-hydroxy-tryptophan. 5-hydroxy-indole. and DOPA (3,4-dihydroxy-phenyI-alanine) was tested in the Fe-induced lipid peroxidation of liver microsomes of normal- and vitamin E-deficient rats, using ascorbate as a reductant. Lipid peroxidation was monitored as low-level chemilu-minescence, indicative of generation of electronically-excited states arising from the recombination of secondary lipid peroxyl radicals.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

How to Characterize a Biological Antioxidant

An antioxidant is a substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. Many substances have been suggested to act as antioxidants in vivo, but few have been proved to do so. The present review addresses the criteria necessary to evaluate a proposed antioxidant activity. Simple methods for assessing the possibility of physiologically-feasible scavenging of important biological oxidants (superoxide, hydrogen peroxide, hydroxyl radical, hypochlorous acid, haem-associated ferryl species, radicals derived from activated phagocytes, and peroxyl radicals, both lipid-soluble and water-soluble) are presented, and the appropriate control experiments are described. Methods that may be used to gain evidence that a compound actually does function as an antioxidant in vivo are discussed. A review of the pro-oxidant and anti-oxidant properties of ascorbic acid that have been reported in the literature leads to the conclusion that this compound acts as an antioxidant in vivo under most circumstances.     

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

Effects of diet and activity on lipid levels of adult Mediterranean fruit flies

Abstract. The objectives of this study were to determine whether adult Mediterranean fruit flies, Ceratitis capitata Wiedeman (Diptera: Tephritidae), are capable of synthesizing lipids, and whether adult diet affects this ability.Lipid levels in females fed protein and carbohydrate or carbohydrate alone declined significantly from emergence to the fourth day of life and then rose back to teneral levels on the fifth day, before oviposition took place on the sixth day.In males fed protein and carbohydrate, lipid levels initially declined as males aged and then stabilized.In carbohydrate-fed males lipid levels declined following emergence and recovered somewhat by the sixth day.Lipid levels declined significantly when flies underwent post-emergence starvation, but after substantial feeding on the above-mentioned diets they eventually (within 6–7 days) reached teneral levels in all experimental groups.Multivariate analysis of variance revealed that differences in lipid contents are primarily related to the flies' age, which corresponds to the various sex-specific activities these flies exhibit.Average lipid investment in eggs was found to equal teneral lipid levels in females.Without lipogenic abilities, oviposition would completely deplete female lipid reserves.We conclude that adult medflies are capaple of lipid synthesis, and that this capability is modulated by individual and sex-specific activity patterns.     

Toxicity to alveolar macrophages in rats following parenteral injection of mercuric chloride

Alveolar macrophages collected by pulmonary lavage from male Fisher-344 rats at intervals (24–72 h) after HgCl₂ injection (1–5 mg/kg, sc) were analyzed by several techniques. Within 24–72 h, the macrophages showed morphological signs of activation (hypertrophy and ruffled plasma membrane). Lipid peroxidation (increased malondialdehyde concentration) was not detected until 48 h. Dose- and time-related effects of HgCl₂ on malondialdehyde concentration and time-related effects of HgCl₂ on malondialdehyde concentration and mercury content of alveolar macrophages were observed 24–72 h postinjection. Diminished cell viability occurred only at 72 h after the highest dosage of HgCl₂. This study demonstrates that the alveolar macrophage was a cellular target for mercury toxicity following parenteral exposure to HgCl₂.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Hydrogen Peroxide and the Proliferation of Bhk-21 Cells

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H₂O₂, such as the superoxide dismutase mimic, copper II—(3,5-diisopropylsalicylate)₂, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 μM exogenous H₂O₂ to the culture medium. Under these conditions, however, intracellular levels of H₂O₂ are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H₂O₂ and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H₂O₂, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to “trigger” their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H₂O₂