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Polyclonal B-cell-activating factors produced by spleen cells of mice stimulated with a cell homogenate of Trypanosoma gambiense.

The effect of injection of a cell homogenate of Trypanosoma gambiense into mice on the production of soluble factors responsible for the induction of polyclonal B-cell activation (PBA) by their spleen cells was examined. PBA was induced by injection of the cell homogenate and was also detected in mice treated with either the serum or the spleen cells of mice treated with the cell homogenate. PBA-inducing activity became detectable in the serum and spleen cells as early as 12 h after injection of the cell homogenate, reached a peak on day 2, and then decreased. This activity was also detected in the culture medium of spleen cells obtained 2 days after injection of the cell homogenate. For determination of the type of spleen cells producing the PBA-inducing factor, the day 2 spleen cells were fractionated on the basis of differences in their adhesive properties. Spleen cells in the effluents from a Sephadex G-10 column (T and B cells) and a nylon wool column (T cells) and those adhering to a plastic flask (macrophages) all produced the PBA-inducing factor. The production of PBA-inducing factor by whole spleen cells and by cells adherent to the plastic flask was not affected by treatment with anti-Thy-1.2 antibody and complement. These data suggest that soluble factors derived from macrophages and T cells could contribute to the induction of PBA. The PBA-inducing activity in the conditioned medium was completely inactivated by treatment at pH 2.0, heating at 56 degrees C for 30 min, or exposure to 0.1% sodium dodecyl sulfate. The results are discussed in relation to cytokines that could affect B-cell activation.

Animals↗

Reduced cell yields of mouse cell line cultures after exposure to homogenates of multiple sclerosis tissues.

The total cell yields in cultures of a mouse cell line termed "PAM" were reduced markedly after exposure to multiple sclerosis (MS) homogenates. The reductions were noted as early as the second subcultivation. The effect was produced by eight of eight MS brain homogenates and three of three MS spleen homogenates; the samples were from 10 MS cases. The reduction in total cell yields (compared to medium-treated cultures) was not observed after exposure to homogenates, six brain and two spleen, from eight non-MS cases. The effect was produced by the 50-nm filtrate of a pool of MS brain homogenates. Five of the MS-inoculated cultures were maintained for 18 subcultivations, and the reduction in total cell yields persisted throughout the series. The capacity to induce the reduction in cell yields was present in cell-free lysates from five of five MS cultures at passage 18, as was the capacity to cause a decrease in the polymorphonuclear neutrophils in the peripheral blood of mice. Calculation of the dilution effect (at least 10(18)-fold) that had occurred by the end of the passage series proved that the factor(s) causing the cell change in culture and the polymorphonuclear neutrophil change in mice had replicated in PAM cells.

Animals↗

Differential biotransformation of glyceryl trinitrate by red blood cell-supernatant fraction and pulmonary vein homogenate.

We have demonstrated previously that glyceryl trinitrate (GTN) undergoes biotransformation to two glyceryl dinitrate (GDN) metabolites in the human red blood cell-supernatant fraction (RBC-SF) by hemoglobin-mediated and sulfhydryl-dependent enzymatic mechanisms. In the present study, we have shown that biotransformation of GTN in rabbit RBC-SF yields a glyceryl-1,2-dinitrate (1,2-GDN)/glyceryl-1,3-dinitrate (1,3-GDN) ratio of 5.3. Following inhibition of hemoglobin-mediated biotransformation of GTN by carbon monoxide (CO), the 1,2-GDN/1,3-GDN ratio was 2.1. Following inhibition of sulfhydryl-dependent biotransformation by N-ethylmaleimide (NEM), the 1,2-GDN/1,3-GDN ratio was 30.0. We have demonstrated previously that for GTN-induced vasodilation of isolated bovine pulmonary vein (BPV), the 1,2-GDN/1,3-GDN ratio was 7.1, which indicated that a hemoprotein-dependent process was involved in GTN biotransformation. To determine if this was the case, the biotransformation of GTN (0.51 microM) was studied in BPV homogenates; 31.1 pmol GDN/mg BPV protein was formed in 20 min. The 1,2-GDN/1,3-GDN ratio was 1.1, which indicated that hemoprotein-mediated biotransformation did not occur. This conclusion was supported by the fact that CO did not inhibit GTN biotransformation. GTN biotransformation by BPV homogenate was inhibited 62% by NEM, 89% by boiling of the homogenate, and almost completely by boiling plus NEM. These results indicated that biotransformation of GTN by the BPV homogenate involved in a combination of enzymatic and nonenzymatic processes that were mostly sulfhydryl dependent. It is concluded that the mechanism for GTN biotransformation in isolated intact BPV, which yielded preferential formation of 1,2-GDN, was rendered nonfunctional upon tissue homogenization.

Animals↗

Sex differences in repolarization homogeneity and its circadian pattern.

The reason for sex differences in arrhythmic risk remains unclear. Heterogeneity of ventricular repolarization is directly linked to arrhythmogenesis; thus we investigated repolarization homogeneity and its circadian pattern in men and women. During 24-h Holter recordings in 60 healthy subjects (27 males), a 12-lead electrocardiogram (ECG) was obtained every 30 s. RR and QT intervals, and, after singular-value decomposition, two characteristics of repolarization homogeneity were calculated in each ECG. Corrected QT (QTc) values were obtained using an individually optimized heart rate (HR) correction formula. All values were averaged over 10-min time bands from 0000 to 2400. There were substantial sex differences in both global repolarization homogeneity (measured by the total cosine of the angle between QRS and T wave vectors) and regional homogeneity of repolarization (quantified independently by the relative T wave residuum). Whereas women throughout the 24 h followed more closely the pattern of inverse sequence between depolarization and repolarization, they also showed much higher localized repolarization heterogeneity than men. In both women and men, repolarization irregularity was greatest during morning hours. A sex difference was also observed for HR and QTc interval; however, the circadian patterns of the repolarization homogeneity descriptors were different from those of HR and QTc intervals.

Adolescent↗

Determination of human skeletal muscle buffer value by homogenate technique: methods of measurement.

The human muscle buffer value (beta) is most frequently determined by either fixed acid titration of a homogenate ["in vitro" beta (beta vit)] or measurement of the change in lactate concentration (delta [La]) relative to the change in muscle homogenate pH after high-intensity exercise ["in vitro" beta = - delta [La]/delta pH (beta viv)]. We sought to compare beta viv, determined after isometric and dynamic exercise to exhaustion (approximately 60 s), with beta vit. Resting (R) and postexercise (E) biopsy samples were taken from vastus lateralis muscles of 43 human volunteers. Freeze-dried muscle was homogenized (30 mg/ml) in NaF (0.01 M) for the measurement of muscle pH (R and E). beta vit was determined by HCl (0.01 M) titration of the homogenate over the pH range 7.1-6.5. Muscle lactate was measured by enzymatic assay. There was no significant difference between beta viv determined after isometric (n = 35) or dynamic (n = 8) exercise to fatigue (170 vs. 168 mmol H+.kg dry muscle mass-1.pH-1, respectively; P > 0.05). Values for beta vit in the corresponding muscle samples (R) were approximately 7-8% lower (156 +/- 25 vs. 157 +/- 18 mmol H+.kg dry muscle mass-1.pH-1, respectively). There was no significant difference (P = 0.278) between the measured decline in muscle homogenate pH after exercise and the reduction in pH predicted from beta vit and delta [La], indirectly confirming the lack of any significant difference between beta viv and beta vit.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Reduced activity of acetylcholinesterase in canine tracheal smooth muscle homogenates after active immune-sensitization.

Previous investigations have suggested that immune-sensitization increases airway smooth muscle responsiveness to cholinomimetic stimulation by reducing the rate of degradation of acetylcholine. To examine the hypothesis that increased cholinomimetic responsiveness of tracheal smooth muscle (TSM) caused by immune-sensitization results from inhibition of acetylcholinesterase (AChase) activity, we developed a method for direct measurement of AChase activity in homogenates of TSM obtained from mongrel dogs actively sensitized in vivo to ragweed pollen extract (n = 7) and sham-sensitized littermate controls (n = 7). For both sensitized and control specimens, saturation of AChase was obtained at approximately 3.12 mM substrate (acetylthiocholine); however, maximal enzyme activity in homogenates of ragweed-sensitized tissues was significantly less (0.862 +/- 0.088 absorbance units/min/mg protein [AU/min/mg]) compared to control homogenates (1.590 +/- 0.129 AU/min/mg; P less than 0.001). Kinetic analysis (Eadie-Hofstee plot) indicated similar Michaelis constants (Km) for AChase from ragweed-sensitized (0.360 +/- 0.063) and control (0.336 +/- 0.062) homogenates (P = NS). The concentration of physostigmine eliciting half-maximal inhibition (Ki) of AChase activity also was similar for tissues from sensitized (-7.92 +/- 0.032 log M) and control animals (-7.86 +/- 0.012 log M; P = NS). Pretreatment with selected mediators of anaphylaxis (10(-4) M histamine, 10(-6) M serotonin, 10(-5) M prostaglandin E2, 10(-6) M prostaglandin F2 alpha, and 10(-7) M leukotriene D4) did not affect AChase activity. Our data demonstrate reduced AChase activity in homogenates of canine TSM after active immune-sensitization in vivo. This corresponds to functional augmentation of cholinomimetic contraction in actively sensitized tissues.

Acetylcholinesterase↗

Speckle reduction with edge preservation in medical ultrasonic images using a homogeneous region growing mean filter (HRGMF).

In this paper, we propose a new post formation adaptive image filtering technique, to be called the homogeneous region growing mean filter, in order to reduce speckle noise with edge preservation in medical ultrasonic images. First, to find a proper seed region, an initially assumed seed region, which is larger than the average speckle size around a given filtering point, is successively contracted according to a certain local homogeneity criterion. Once the seed region is determined, the next step is to grow the homogeneous region successively based on some measures of local homogeneity and similarity of the neighboring region. The output of the proposed filter for each filtering point is obtained from the arithmetic mean of the grown locally homogeneous region. Several simulation results are presented to illustrate the performance of the proposed filter. They show that the proposed technique effectively smoothes ultrasonic speckle and completely suppresses isolated impulsive noise over the entire texture in addition to preserving the edge information.

Algorithms↗

Elevated tissue factor levels in leukemic cell homogenate.

Tissue factor (TF) antigen and activity were measured in leukemic cell homogenates. In leukemic cell homogenate, especially that of acute promyelocytic leukemia (APL), both TF antigen and activity were significantly higher than these levels in the mononuclear cells obtained from healthy volunteers. Both TF antigen and activity were significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. In leukemic cell homogenates, there was a close correlation between TF antigen and TF activity. The TF activity/TF antigen ratio was significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. As the TF activity was not increased in lymphocytic leukemia cell homogenates to which were added phospholipids, the decrease in TF activity in lymphocytic leukemia might not be due to phospholipid in the leukemic cell membrane. Values for TF activity, TF antigen, and the TF activity/TF antigen ratio in leukemic cell homogenate from patients with disseminated intravascular coagulation (DIC) were significantly higher than those in patients without DIC. Therefore, the measurement of TF antigen and activity in leukemic cells could be useful for the prediction of DIC.

Antigens, Neoplasm↗

Magnetic stimulation for non-homogeneous biological structures.

BACKGROUND: Magnetic stimulation has gained relatively wide application in studying nervous system structures. This technology has the advantage of reduced excitation of sensory nerve endings, and hence results in quasi-painless action. It has become clinically accepted modality for brain stimulation. However, theoretical and practical solutions for assessment of induced current distribution need more detailed and accurate consideration. Some possible analyses are proposed for distribution of the current induced from excitation current contours of different shape and disposition. Relatively non-difficult solutions are shown, applicable for two- and three-dimensional analysis. METHODS: The boundary conditions for field analysis by the internal Dirichlet problem are introduced, based on the vector potential field excited by external current coils. The feedback from the induced eddy currents is neglected. Finite element modeling is applied for obtaining the electromagnetic fields distribution in a non-homogeneous domain. RESULTS: The distributions were obtained in a non-homogeneous structure comprised of homogeneous layers. A tendency was found of the induced currents to follow paths in lower resistivity layers, deviating from the expected theoretical course for a homogeneous domain. Current density concentrations occur at the boundary between layers, suggesting the possibility for focusing on, or predicting of, a zone of stimulation. CONCLUSION: The theoretical basis and simplified approach for generation of 3D FEM networks for magnetic stimulation analysis are presented, applicable in non-homogeneous and non-linear media. The inconveniences of introducing external excitation currents are avoided. Thus, the possibilities are improved for analysis of distributions induced by time-varying currents from contours of various geometry and position with respect to the medium.

Electric Stimulation↗

Detection of cytokine protein expression in mouse lung homogenates using suspension bead array.

BACKGROUND: The objective for this present study was to determine whether or not suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates. Here, we report on suspension bead array as a feasible method for detection of lipopolysaccharide (LPS)-evoked changes in cytokine protein expression in mouse lung tissue homogenates. MATERIALS AND METHODS: Mice were treated (0.2 ml, intraperitoneal, i.p.) with phosphate buffered saline (PBS) or LPS (0.25 mg/ml) and sacrificed at either 2- or 24-hours post treatment. Formalin-fixed and paraffin-embedded lung sections were evaluated by light microscopy. Flash frozen lung tissues were homogenized for measurement of various cytokine protein expression levels using suspension bead array, antibody array and ELISA. Comparison between groups was performed using a Wilcoxon signed rank test. RESULTS: Pulmonary perivascular edema and an accumulation of mixed cell infiltrates within blood and lymphatic vessels, as well as in the adjacent interstitium, were present at both 2- and 24-hours following LPS treatment. A minimal increase in the number of alveolar macrophages was also observed in the 24-hour LPS-treated mice only. The suspension bead array assay revealed statistically significant increases in mouse lung tissue homogenate levels of interleukin-6 (IL-6) and granulocyte/macrophage colony-stimulating factor (GM-CSF) proteins and a decrease in IL-2 protein at 24-hours post LPS-treatment only. Similar cytokine protein expression patterns were observed using antibody array. Significantly increased IL-6 protein expression levels were also detected using ELISA, which correlated with the suspension bead array data. CONCLUSION: The present study shows that suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates.

Journal Article↗

Localization of estrogen synthetase in the chorionic villus fraction after homogenization of human term placenta.

In previous investigations of human placental estrogen synthetase (aromatase), activity was found in the microsomal and mitochondrial fractions after homogenization and subcellular fractionation of placental tissue in isotonic sucrose. We report here that the majority of the placental aromatase sediments in a much heavier particulate fraction than the microsomal or mitochondrial fractions. After low speed centrifugation of the placental homogenate, only about 20% of the homogenate aromatase activity was measured in the supernatant, the precursor of the microsomal and mitochondrial fractions. Even after extensive homogenization in high speed shearing-type blenders and tight-fitting glass homogenizers the majority of the placental aromatase (more than 70%) consistently sedimented at very low centrifugal forces (100 x g ro 10 min) independent of whether the suspending medium contained up to 1.2M sucrose, or IM NaCl, or 50% glycerol. By selectively separating components of the low speed pellet via a sieving procedure or manual dissection of placental tissue, we found that the majority of the enzyme activity is associated with placental chorionic villus fragments. Several extensively washed preparations of villus fragments with aromatase specific activities similar to that found in placental microsomes were synthesis in the villus aromatase fraction was determined to be 1beta,2beta-H removal which is identical with that by placental microsomes and human ovaries. Evidence for the presence of cytochrome P-450 in the villus fraction was obtained by measuring the carbon monoxide-dithionite difference spectrum.

Androstenedione↗

[Effect of idebenone and various nootropic drugs on lipid peroxidation in rat brain homogenate in the presence of succinate].

The effects of idebenone and various nootropic drugs on lipid peroxidation in rat brain homogenate were examined. Idebenone inhibited lipoperoxide (LPO) production in brain homogenate in a concentration-dependent manner, with an IC50 of 38 microM. The inhibition was strongly enhanced (about 100-fold) by adding succinate, a substrate in the mitochondrial respiration. The optimal concentration of succinate was 0.5 mM. Inhibition of lipid peroxidation in brain homogenate by various nootropic drugs in the presence or absence of succinate was then examined. Drugs added to the brain homogenate at 100 microM in the absence of succinate inhibited LPO production in the order: idebenone greater than vinpocetine greater than bifemelane greater than indeloxazine greater than calcium hopantenate. However, when the drugs were added at 1 microM in the presence of succinate, only idebenone demonstrated inhibition. These results suggest that although almost all of the drugs tested inhibit lipid peroxidation in brain homogenate, only idebenone is activated by succinate, the other drugs being insensitive to this compound.

Animals↗

A new liquid homogeneous assay for HDL cholesterol determination evaluated in seven laboratories in Europe and the United States.

We evaluated a new liquid homogeneous assay for the direct measurement of high density lipoprotein cholesterol (HDL-C Plus) in seven laboratories. The assay includes two reagents which can be readily used in most available clinical chemistry analyzers. The total CVs of the new method were below 4.6% and the bias in relation to the designated comparison method was below 3.9%. The total error ranged between 4 to 7%. HDL-C values determined by this method were in good agreement with those obtained by the old homogeneous assay using lyophilized reagents, and other homogeneous and precipitation assays (0.944 < r < 0.996). The assay was linear up to at least 3.89 mmol/l HDL-C. Hemoglobin did not interfere, whereas in icteric samples slight deviations were observed. Lipemia up to 11.3 to 22.6 mmol/l triglycerides did not interfere with this homogeneous HDL-C assay. In samples of patients with paraproteinemia, discrepant results were seen. This liquid homogeneous HDL-C assay was easy to handle and produced similar results in all laboratories participating in this study. This method will enable clinical laboratories to reliably measure HDL-C for risk assessment of coronary heart disease.

Artifacts↗

Reproduction in rats and mice isoimmunized with homogenates of ovary or testis with epididymis, or sperm suspensions.

Female rats and mice were injected with homologous spermatozoa without adjuvant, or with homogenates of ovary or testis+ epididymis in adjuvant. Ovulation was not inhibited and the numbers of eggs released were normal. Fertilization rates were significantly reduced in the mouse by all tissues injected and in the rat by injection of ovarian homogenate only. Fracture of and a weak precipitate on the zona pellucida of the eggs were observed in the mouse but not in the rat after injection of ovarian homogenates. The number of pregnancies and litter sizes were reduced following the first and second mating after treatment in the mouse but not in the rat. When eggs with or without follicular cells were examined in vitro, fertilizability was very low if the donor had been treated with ovarian homogenate, better with testis+epididymis homogenate and best after treatment with sperm suspension. Failure of fertilization appeared to be the major cause of infertility after such treatments.

Animals↗

The occurrence of pituitary prolactin depletion-transformation in lactating rats: dependence on strain of rats, homogenization conditions and method of assay.

The objectives of this study were to determine whether pre-release transformation (depletion) of pituitary prolactin occurs as the result of suckling to the same extent in several strains of lactating rats, the molecular nature of the transformed hormone, whether the quantity of transformed (depleted) prolactin recovered is dependent upon the type of homogenization buffer used and whether the method of assay influences the extent to which transformed prolactin is detected. During the course of these experiments other factors such as the methods of handling and storing pituitaries and homogenates were also found to influence the amount of prolactin recovered. The results indicated that transformation of prolactin is a very labile event which is affected by many factors. Strain and supplier of rats was critical to the observation of suckling-induced depletion of prolactin, with Spartan- and Holtzman-derived Sprague-Dawley strains exhibiting the most consistent responses. When transformation was observed, it mattered little which buffer was used for homogenization; however, alkaline or acidic buffers extracted more prolactin than did neutral buffers. Triton X-100 also significantly enhanced the efficiency of extraction by neutral buffers. Maintaining pituitaries on dry ice immediately upon removal from the animal increased the amount of prolactin recovered, as did freezing the homogenate for 1-5 weeks before assay. The addition of the protease inhibitor, benzamidine hydrochloride, did not affect the pituitary content of prolactin. Assay of prolactin by polyacrylamide electrophoresis and densitometry yielded more prolactin than either radioimmunoassay or the Nb2 lymphoma bioassay. The molecular nature of pituitary prolactin, extracted at neutral pH, as judged by gel filtration was altered slightly but consistently by suckling, such that large molecular forms increased at the expense of the smallest molecular form. We conclude from these studies that great care must be exercised when attempting to characterize dynamic changes in pituitary prolactin content in lactating rats. Strain and supplier of rats, methods of handling and storing pituitaries, types of buffers used for homogenization and methods of assay all influence the amount of prolactin recovered and can influence the extent to which rapid changes in pituitary prolactin are detected.

Animals↗

Predicting the effects of habitat homogenization on marine biodiversity.

Seafloor habitats throughout the world's oceans are being homogenized by physical disturbance. Even though seafloor sediments are commonly considered to be simple and unstructured ecosystems, the negative impacts of habitat homogenization are widespread because resident organisms create much of their habitat's structure. We combine the insight gained from remote sensing of seafloor habitats with recently developed analytical techniques to estimate species richness and assess the potential for change with habitat homogenization. Using habitat-dependent species-area relationships we show that realistic scenarios of habitat homogenization predict biodiversity losses when biogenic habitats in soft sediments are homogenized. We develop a simple model that highlights the degree to which the reductions in the number of species and functional diversity are related to the distribution across habitats of habitat-specific and generalist species. Our results suggest that, by using habitat-dependent species-area relationships, we can better predict variation in biodiversity across seafloor landscapes and contribute to improved management and conservation.

Biodiversity↗

Homogenization dynamics and introduction routes of invasive freshwater fish in the Iberian Peninsula.

Nonnative invasive species are one of the main global threats to biodiversity. The understanding of the traits characterizing successful invaders and invasion-prone ecosystems is increasing, but our predictive ability is still limited. Quantitative information on biotic homogenization and particularly its temporal dynamics is even scarcer. We used freshwater fish distribution data in the Iberian Peninsula in four periods (before human intervention, 1991, 1995, and 2001) to assess the temporal dynamics of biotic homogenization among river basins. The percentage of introduced species among fish faunas has increased in recent times (from 41.8% in 1991 to 52.5% in 2001), leading to a clear increase in the similarity of community composition among basins. The mean Jaccard's index increase (a measure of biotic homogenization) from the pristine situation to the present (17.1%) was similar to that for Californian fish but higher than for other studies. However, biotic homogenization was found to be a temporally dynamic process, with finer temporal grain analyses detecting transient stages of biotic differentiation. Introduced species assemblages were spatially structured along a latitudinal gradient in the Iberian Peninsula, with species related to sport fishing being characteristic of northern basins. Although the comparison of fish distributions in the Iberian Peninsula and France showed significant and generalized biotic homogenization, nonnative assemblages of northeastern Iberian basins were more similar to those of France than to those of the rest of the Iberian Peninsula, indicating a main introduction route. Species introduced to the Iberian Peninsula tended to be mainly piscivores or widely introduced species that previously had been introduced to France. Our results indicate that the simultaneous analysis of the spatial distribution of introduced assemblages (excluding native species that reflect other biogeographical patterns) and their specific traits can be an effective tool to detect introduction and invasion routes and to predict future invaders from donor regions.

Animals↗

Tyrosinase-induced phenoxyl radicals of etoposide (VP-16): interaction with reductants in model systems, K562 leukemic cell and nuclear homogenates.

Etoposide (VP-16) is an antitumor drug currently in use for the treatment of a number of human cancers. Mechanisms of VP-16 cytotoxicity involve DNA breakage secondary to inhibition of DNA topoisomerase II and/or direct drug-induced DNA strand cleavage. The VP-16 molecule contains a hindered phenolic group which is crucial for its antitumor activity because its oxidation yields reactive metabolites (quinones) capable of irreversible binding to macromolecular targets. VP-16 phenoxyl radical is an essential intermediate in VP-16 oxidative activation and can be either converted to oxidation products or reduced by intracellular reductants to its initial phenolic form. In the present paper we demonstrate that the tyrosinase-induced VP-16 phenoxyl radical could be reduced by ascorbate, glutathione (GSH) and dihydrolipoic acid. These reductants caused a transient disappearance of a characteristic VP-16 phenoxyl radical ESR signal which reappeared after depletion of the reductant. The reductants completely prevented VP-16 oxidation by tyrosinase during the lag-period as measured by high performance liquid chromatography; after the lag-period VP-16 oxidation proceeded with the rate observed in the absence of reductants. In homogenates of human K562 leukemic cells, the tyrosinase-induced VP-16 phenoxyl radical ESR signal could be observed only after a lag-period whose duration was dependent on cell concentration; VP-16 oxidation proceeded in cell homogenates after this lag-period. In homogenates of isolated nuclei, the VP-16 phenoxyl radical and VP-16 oxidation were also detected after a lag-period, which was significantly shorter than that observed for an equivalent amount of cells. In both cell homogenates and in nuclear homogenates, the duration of the lag period could be increased by exogenously added reductants. The duration of the lag-period for the appearance of the VP-16 phenoxyl radical signal in the ESR spectrum can be used as a convenient measure of cellular reductive capacity. Interaction of the VP-16 phenoxyl radical with intracellular reductants may be critical for its metabolic activation and cytotoxic effects.

Ascorbic Acid↗