Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Dopamine receptor elevation by cholecystokinin

High concentrations of cholecystokinin (CCK) in the striatum and limbic areas of the brain suggest that this peptide may influence dopaminergic transmission. Thus, the effect of CCK on dopamine D₂ receptors in the striatum and nucleus accumbens of the rat brain both in vitro and in vivo (central and peripheral administration) was studied by determining the binding of ³H-spiperone. The density (B_max) of D₂ receptors was elevated (a) by 20% in the accumbens upon in vitro co-incubation with 10⁻⁶ M CCK. (A non-significant drop of 10% occurred in the striatum); (b) by about 40% in the accumbens and 25% in the striatum after continuous intraventricular infusion of CCK for 24 hr. The increase in receptor density in the accumbens was maintained for 14 days and in both tissues was specific to CCK (neurotensin infusion did not alter ³H-spiperone binding); (c) by 20% in the accumbens and 15% in the striatum 3 hr after a single IP injection of 50 μg/kg CCK or caerulein, and maintained up to 14 days later. These results suggest that CCK elevates dopamine D₂ receptors in the accumbens and striatum and may be a physiological modulator of the dopaminergic system.     

Effect of the proportions of neutral detergent fibre and starch, and their degradation rates, on in vitro ruminal fermentation

Effects of proportions of neutral detergent fibre (aNDFom) and starch, as well as their degradation rates, on rumen fermentation were tested using an in vitro rumen simulation system (SIMCO). The in vitro system was designed to simulate selective particle retention and had an average fluid volume of 1150 ml with a liquid dilution rate of approximately 0.07 h⁻¹. Two types of hay (aNDFom sources) and two types of starch were each included at two different levels in the diet and were examined in an experiment following a 2×2×2 factorial arrangement of treatments (eight diet combinations). The hay was either late-cut timothy (Phleum pretense L.) or early cut meadow grass (Poa pratensis L.), with ruminal in situ aNDFom digestion rates of 0.03–0.04 and 0.07–0.08 h⁻¹, respectively. The two starch types were raw (R) and cooked (C) potato starch with previously determined in vitro ruminal digestion rates of 0.04 and 0.20 h⁻¹, respectively. The starch levels were 300 and 600 g/kg diet dry matter (DM) with the remaining being hay (282–682 g/kg DM) and peptone (14–111 g/kg DM). The aNDFom level varied among the diets with different starch levels and hay types. The peptone acted as a source of peptides and, together with ammonia salts from buffer, was used to balance the N contents of the diets. The feeding level for each of the eight vessels was 28 g DM/d. Two 10-day simulations were made with the system. The average pH was higher (P<0.05) for all treatments with raw potato starch (6.19) versus cooked starch (6.07). Protozoa scores, on a qualitative scale, declined faster at the higher starch level. The aNDFom digestibility was, as expected, higher (P<0.001) for meadow hay (0.57) than timothy (0.32), and was also higher (P<0.001) at the lower starch level (0.54) versus the higher (0.35). Microbial protein production efficiency (mg microbial N/g organic matter truly digested) was higher for the faster degrading aNDFom (P<0.01) and starch (P<0.05) sources, but was not affected by starch level. Cooked starch resulted in a lower acetate proportion (449 mmol/mol versus 591 mmol/mol VFA; P<0.001) but higher proportions of propionate (297 mmol/mol versus 236 mmol/mol VFA; P<0.001), and butyrate (169 mmol/mol versus 127 mmol/mol VFA; P<0.01). Butyrate increased with starch level (127 mmol/mol versus 169 mmol/mol VFA; P<0.01), and was also higher for meadow hay versus timothy (168 mmol/mol versus 128 mmol/mol VFA; P<0.01). Interactions between the treatments demonstrate that the response in VFA pattern to starch level is dependent on starch and aNDFom sources. Substrates such as starch and aNDFom are fermented differently depending on their rates of ruminal degradation.     

非洲菊原生质体制备及瞬时转化系统的建立

非洲菊(Gerbera hybrida)已成为研究复杂花序进化与发育的模式植物。以非洲菊无菌组培苗叶片为材料,分析不同浓度纤维素酶及离析酶配比对非洲菊原生质体提取效率的影响,建立了稳定、高效的非洲菊原生质体提取与瞬时转化体系。结果表明,以2.0%纤维素酶+0.3%离析酶组合,在0.4 mol·L~(–1)甘露醇溶液中,酶解4小时,酶解温度为25°C,得到产量高达2.2×10~7个·g~(–1) FW及活力较高的原生质体,可直接用于植物蛋白亚细胞定位和蛋白间相互作用实验,转化效率达80%。该研究建立了非洲菊原生质体制备和瞬时转化体系,为非洲菊基因功能研究奠定重要的技术基础。     

Peripheral oxytocin treatment modulates central dopamine transmission in the mouse limbic structures

The effects of subcutaneous (s.c.) oxytocin treatment have been investigated on various parameters of dopaminergic neurotransmission in basal forebrain structures (nucleus olfactorius posterior + nucleus accumbens + septum) of the mouse. Acute oxytocin treatment failed to influence dopamine utilization in the basal forebrain. Following chronic injections of oxytocin (0.2 mg/kg) for 8 8 days, the neuropeptide decreased dopamine utilization. Neither in vivo nor in vitro oxytocin treatment was capable of influencing the in vitro uptake of [³H]dopamine in basal forebrain slices. The spontaneous release of [³H]dopamine (in the presence of 4.2 mM K⁺) from basal forebrain tissue slices was not affected by in vitro or acute or chronic in vivo oxytocin treatment. The stimulated release of [³H]dopamine (in the presence of 30 mM K⁺) was significantly inhibited by chronic in vivo oxytocin administration. Chronic oxytocin treatment decreased the B_max value of [³H]spiroperidol binding in the basal forebrain. The dissociation constant (K_d) of [³H]spiroperidol binding was not influenced by oxytocin. The data indicate that peripheral oxytocin treatment is capable of modifying dopaminergic neurotransmission in mouse basal forebrain regions.     

In vitro enzyme evolution: the screening challenge of isolating the one in a million

The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10⁴–10⁷ mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.     

Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca^2 + also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca^2 + oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca^2 + oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca^2 + oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of E - C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca^2 + oscillation. The intercellular synchronization of intracellular Ca^2 + oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca^2 + oscillation, but possibly contributed to the stability of the oscillatory rhythm.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Enkephalin analogs and dermorphin in the conscious dog: Structure-activity relationships

Those structural features of enkephalins (ENK) responsible for in vitro organ bath and receptor binding activity have been investigated in detail in the conscious, chronically instrumented dog. Amide analogs of Leu⁵-ENK display reduced activity, which is restored by D-Ala² substitutions. N-terminal L-Tyr is required for full opiate activity. Although proven δ-receptor agonists do appear generally more active, distinctions made in vitro between μ and δ binding are not apparent in the complex hemodynamic responses which occur in the intact unanesthetized dog. The amphibian skin peptide dermorphin, which contains D-Ala², elevates heart rate, systemic arterial pressure, and induces vomiting with near maximal activity at a dose of 1.0 μg/kg; this activity is inhibited by naloxone. This activity, coupled with dermorphin's apparent presence in mammalian tissue, suggests that it may represent another peptide factor in cardiovascular regulation. In the conscious dog, ENK elevate heart rate and systemic arterial pressure; this activity does not appear to be fully explained by in vitro receptor models.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Purine enzyme inhibition fails to alter benzodiazepine receptor binding in brain

Binding of [³H]flunitrazepam to benzodiazepine receptors in brain from several species, including human, was measured in vitro in the presence and absence of purine-metabolizing enzyme inhibitors. Incubation with potent inhibitors of either adenosine deaminase (2′-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)-adenine) or guanine deaminase (5-amino-4-imidazole carboxamide) failed to alter [³H]flunitrazepam binding in homogenates of several different regions of human, rabbit, rat or guinea pig brain. These findings are in contrast to those of Norstrand et al. [Enzyme 29, 61–65 (1983)] who reported substantial alterations in [³H]flunitrazepam binding to human brain membranes in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (increase) and 5-amino-4-imidazole carboxamide (decrease). In our studies, [³H]flunitrazepam binding was also unaltered in more anatomically intact brain sections following treatment with purine enzyme inhibitors. Furthermore, in vivo administration of erythro-9-(2-hydroxy-3-nonyl)-adenine to mice at a dose (200 mg/kg, i.p.) known to almost totally inhibit central adenosine deaminase activity also failed to alter brain [³H]flunitrazepam binding measured ex vivo, 30–120 min post injection.

While previous studies have shown that purines such as inosine interact with benzodiazepine receptors, our results raise some questions about the role of endogenous purines in regulating benzodiazepine receptors, at least in vitro and also acutely _vivo following purine enzyme inhibitor administration.     

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4°C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765–768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (Pl) fraction and cold-soluble tubulin in the supernatant (Sl) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of ³²P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of ³²P.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.     

Rapid increase in serum lipid peroxide 4-hydroxynonenal (HNE) through monocyte NADPH oxidase in early endo-toxemia

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.     

The digestion of dietary protein bound by condensed tannins in the gastro-intestinal tract of sheep

The digestion of dietary protein bound by condensed tannins (CTs) in ruminants was investigated by determining the extent of dissociation of insoluble ¹²⁵I-BSA + CT complexes administered to abomasally and intestinally fistulated sheep. The extent of dissociation was registered as the true digestibility of iodinated bovine serum albumin (¹²⁵I-BSA). The true digestibility of ¹²⁵I-BSA originally bound to Leucaena pallida CT (0.721) was lower (P<0.05) than that of ¹²⁵I-BSA originally bound to L. leucocephala CT (0.880) between the abomasum and terminal ileum. These results indicate that differences in the ability of CT to inhibit ¹²⁵I-BSA digestion in vivo matched the relative abilities of the same CT to bind BSA in vitro, indicating that the in vitro BSA-binding assay for ranking CT behaviour was biologically relevant in vivo. Furthermore, the true digestibility of CT-bound ¹²⁵I-BSA between the mouth and faeces permitted the prediction of the quantitative contribution that CT-bound dietary proteins make to improved nitrogen supply to the small intestines.     

Comparison Between Different Assays for Superoxide Dismutase-Like Activity

The direct and indirect methods for assaying the superoxide dismutase activity of a compound are compared. With the use of a direct method. the mechanism of the catalysis of O₂-dismutation by the tested compound can be determined. while with the indirect method it cannot. and this may lead to misinterpretation of the results. Assuming that the catalysis occurs via the 'ping-pong' mechanism, both the direct and indirect methods are limited to the determination of values of k_cat ≥ 10⁵M^-1s^-1 and k_cat ≥ 3 × 10⁶M^-1s^-1. respectively. Moreover, many side reactions may occur with the indirect method which may interfere with the measurements. Nevertheless. the indirect method approximates better the in vivo conditions than the direct method, and a tested compound that has high SOD activity using a direct method and low SOD activity using an indirect method. will most probably be a poor SOD mimic in vivo.