Reduction Of Tetrazolium Salt Research Articles

Methyl donor nutrients inhibit the growth of human breast cancer cells

Lipotropes (methyl donor nutrients) are involved in one-carbon metabolism including DNA methylation. Hypermethylation of CpG islands within promoter regions of genes is associated with transcriptional inactivation of the gene. Methyl donors may alter the expression of apoptosis-related genes via alterations in DNA methylation and consequently increase the sensitivity of cancer cells to apoptosis. To investigate the effects of lipotropes on breast cancer cell growth and apoptosis, two estrogen receptor-positive breast cancer cell lines, MCF-7 and T47D, as well as a normal mammary cell line, MCF-10A, were cultured in control, lipotrope-supplemented, tamoxifen (TAM), and lipotrope-supplemented plus TAM treatment media. Cell proliferation was measured by a tetrazolium salt reduction assay. Lipotrope supplementation inhibited the growth of both T47D (P < 0.01) and MCF-7 (P < 0.01) cell lines. All treatment groups, except the control groups in all cell lines, demonstrated morphological changes, as assessed by DAPI staining, consistent with apoptotic cell death. Lipotropes significantly decreased the Bcl-2 protein level, quantified by an enzyme immunometric assay, in the T47D cell line (P = 0.01) but not in the MCF-7 cell line. These results show that lipotrope supplementation may be useful in the development of a nutritional strategy for human breast cancer therapy. Supported by NIH-NCI (# R01CA78179-01).

Establishment of a Novel Intervertebral Disc/Endplate Culture Model: Analysis of an Ex Vivo In Vitro Whole-Organ Rabbit Culture System

Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

ETHANOLIC EXTRACTS OF SOPHORA MOORCROFTIANA SEEDS INDUCE APOPTOSIS OF HUMAN STOMACH CANCER CELL LINE SGC-7901 IN VITRO

The seeds of Sophora moorcroftiana are used in Chinese traditional medicine for the treatment of verminosis, infectious diseases, and anti-inflammation. To investigate the antitumor and induction of apoptosis activity of Sophora moorcroftiana seeds, the ethanolic extracts from the seeds was prepared and added into the culture of human stomach cancer cell line SGC-7901 in vitro. The proliferation and apoptosis of cells treated with the ethanolic extracts were assessed by tetrazolium salt reduction (MTT) assay, fluorescence microscopy, transmission electron microscopy, flow cytometry and agarose gel electrophoresis of DNA fragmentation. The results showed that the growth of SGC-7901 cells was strongly inhibited by the ethanolic extracts at the concentration ranging between 0.31-5.00 mg/ml, and the apoptosis of treated cells was induced at the concentration of 1.25, 2.50, and 5.00 mg/ml in vitro. This suggested that the ethanolic extracts from S. moorcroftiana seeds contain potent antitumor fraction(s) on human stomach cancer SGC-7901 cells.

Colorimetric Assay to Determine In Vitro Antibacterial Activity Against Clinical Isolates: Enhanced Activity in Damaged Chinese Masson Pine Needles

AbstractA colorimetric assay for antibacterial susceptibility testing of clinical isolates (Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae) is described based on the reduction of a novel tetrazolium salt, 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS), in the presence of phenazine methosulfate (PMS) as an electron‐coupling agent. The combination of 200 μg/mL MTS with 25 μmol/L PMS resulted in production of large amounts of formazan within 1 h of exposure. In this setting, fractions extracted from Chinese Masson pine (Pinus massoniana Lamb.) needles damaged by the pine caterpillar Dendrolimus punctatus Walker were found to have enhanced levels of antibacterial activity. These fractions, which were designated “Master”, “Technique”, and “Strength”, were isolated and identified by reverse‐phase C18 cartridge concentration, gel filtration, and affinity chromatography. Two fractions purified from healthy and undamaged needles were designated H1 and H2, respectively. For all test bacteria species. Technique produced the lowest minimal inhibitory concentrations (MICs), ranging from 2 to 32 μg/mL, and H2 produced the highest values, with four of the six MICs being higher than 128 μg/mL We found that the Rmax model fitted the data well in that the r2 ranged between 0.87 and 0.96 (median, 0.92) and no statistically significant deviations from the model were found (P= 0.23). The median coefficient of variation of the log RC50 values and the slope m of the fitted model for all six strains among the replicates were 38 and 41%, respectively. In the course of the investigation, the physiological and functional factors involved in pest damage to plants were also explored. In summary, the MTS‐PMS colorimetric assay has advantages over existing methods for the examination of antibacterial activity, and could be developed further such that it would be suitable for screening new antibiotic molecules.(Managing editor: Ping He)

In vitro and in vivo (cyto)toxicity assays using PVC and LDPE as model materials

The choice for a biomaterial is partly based on the outcome of (cyto)toxicity assays. The rationales behind the selection of certain parameters, such as cell lines, controls, and animals, were evaluated using a positive and a negative control, and one experimental sample designed to induce intermediate toxicity. Extraction and direct contact assays were performed using human epidermal keratinocytes and mouse fibroblasts and mouse epithelial cells. Cell survival was measured with the tetrazolium salt (MTT) reduction assay. In addition, local implantation studies were performed in mice and rats. The positive control induced a high degree of toxicity in all in vitro tests performed, indicating that the toxicity observed in the direct contact assay was due to in situ extraction of toxic components. In the direct contact assay the negative control tested on the mouse fibroblasts resulted in a significant reduction of cell survival. No decrease in cell survival was found using the experimental sample. Subcutaneous implantation studies in mice showed that the positive control material induced a severe degeneration in mice. However, in rats just minimal alterations were noted. The experimental material induced moderate responses only in mice. Our results indicate that the direct contact assay provides limited additional information on the cytotoxicity of materials if certain limitations are not taken into account. For the in vivo implantation assay mice were superior to rats in detecting local toxic responses.

Raloxifene-Induced Myeloma Cell Apoptosis: A Study of Nuclear Factor-κB Inhibition and Gene Expression Signature

Because multiple myeloma remains associated with a poor prognosis, novel drugs targeting specific signaling pathways are needed. The efficacy of selective estrogen receptor modulators for the treatment of multiple myeloma is not well documented. In the present report, we studied the antitumor activity of raloxifene, a selective estrogen receptor modulator, on multiple myeloma cell lines. Raloxifene effects were assessed by tetrazolium salt reduction assay, cell cycle analysis, and Western blotting. Mobility shift assay, immunoprecipitation, chromatin immunoprecipitation assay, and gene expression profiling were performed to characterize the mechanisms of raloxifene-induced activity. Indeed, raloxifene, as well as tamoxifen, decreased JJN-3 and U266 myeloma cell viability and induced caspase-dependent apoptosis. Raloxifene and tamoxifen also increased the cytotoxic response to vincristine and arsenic trioxide. Moreover, raloxifene inhibited constitutive nuclear factor-kappaB (NF-kappaB) activity in myeloma cells by removing p65 from its binding sites through estrogen receptor alpha interaction with p65. It is noteworthy that microarray analysis showed that raloxifene treatment decreased the expression of known NF-kappaB-regulated genes involved in myeloma cell survival and myeloma-induced bone lesions (e.g., c-myc, mip-1alpha, hgf, pac1,...) and induced the expression of a subset of genes regulating cellular cycle (e.g., p21, gadd34, cyclin G2,...). In conclusion, raloxifene induces myeloma cell cycle arrest and apoptosis partly through NF-kappaB-dependent mechanisms. These findings also provide a transcriptional profile of raloxifene treatment on multiple myeloma cells, offering the framework for future studies of selective estrogen receptor modulators therapy in multiple myeloma.

A novel simplified ultra-rapid freezing technique for cryopreservation of tissue slices

Cryopreservation offers the potential to maximize the use and availability of biological materials that have a limited supply. This study demonstrates an enhanced technique for the parallel cryopreservation of a series of liver tissue slices using a tray modeled from aluminium foil and low concentrations of a cryoprotectant. Cooling and warming rates of approximately 2000 and 3900 °C min −1, respectively, were achieved as the thermal capacity of the foil-tray was significantly reduced compared to the aluminium sandwich device introduced by Day et al. [S.H. Day, D.A. Nicoll-Griffith, J.M. Silva, Cryopreservation of rat and human liver slices by rapid freezing, Cryobiology 38 (1999) 154–159]. Additionally, the two critical steps involved in the sandwich approach, i.e., clamping the plates and complete filling of the entire space between the plates with liquid, can be omitted using the foil tray. The viability of the slices was verified by measuring tetrazolium salt reduction capacity, cytosolic enzyme lactate dehydrogenase leakage, and ethoxycoumarin metabolism.

Matrix Metalloproteinase-1 Inhibitor from the Aerial Parts of Viola ibukiana MAKINO

which isbased on reduction of soluble yellow MTT tetrazolium salt[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] to a blue insoluble MTT formazan product bymitochondrial succinic dehydrogenase. After compound orUV treatment, the cells were cultured for indicated days; 20µL of MTT (5 mg/mL) was added to each well, and the cellswere incubated for 4 hrs at 37

Agonist and antagonist dual effect of the cross-linked S19 ribosomal protein dimer in the C5a receptor-mediated respiratory burst reaction of phagocytic leukocytes

To examine the behavior of the crosslinked dimer of S19 ribosomal protein (RP S19), a natural C5a receptor ligand, in the C5a receptor-mediated respiratory burst reaction of monocytes and neutrophils. The respiratory burst reaction of leukocytes was quantitatively observed by continuous spectrophotometric measurement of the reduction of a water-soluble tetrazolium salt, WST-1. The RP S19 dimer induced the respiratory burst of monocytes, but not of neutrophils. Furthermore, in neutrophils, the RP S19 dimer inhibited the reaction induced by C5a, but did not affect the formyl-Met-Leu-Phe-induced reaction. The dimer of a deletion mutant at the C-terminal portion of the RP S19 induced a respiratory burst reaction similar to the one induced by C5a, both in monocytes and neutrophils. Inversely, a chimeric fusion protein between C5a and RPS19, consisting of the addition of the 12 C-terminal amino acid residues of RP S19 after the C-terminal Arg74 of the C5a molecule, behaved similarly to the RP S19 dimer. The RP S19 dimer works as an agonist and antagonist of the C5a receptor in the monocyte and the neutrophil respiratory burst reactions, respectively. The switch moiety between the antagonist and agonist of the RP S19 dimer for the C5a receptor in the induction of respiratory burst of phagocytes localizes at the C-terminal region of RP S19.

Antioxidant and lipid lowering activities of Indian black tea.

Indian black tea; CTC leaf and dust, produced by Tata Tea Limited, Kolkata, (India) was studiedin vitro as potential scavenger of oxygen free radicals. Super oxide anions were generated in a system containing xanthine-xanthine oxidase (enzymic system) and by NADH- phenozine methosulphate (non enzymic system). Anions were assayed in terms of uric acid formation and reduction of nitroblue tetrazolium salt, which were shown to be suppressed by tea extracts. Extracts from both leaf and dust also inhibted the formation of hydroxyl radicalsin vitro in the enzymic system comprising hypoxanthine-Cu(+2)-sodium ascorbate and xanthine oxidase and in non enzymic system of deoxyribose-Cu(+2)-sodium ascorbate and H(2)O(2) as well as the Cu(+2) induced lipid peroxidation in human low density lipoprotein. Feeding with black tea in normal rats for sixty days increased their antioxidant activity and their liver microsomes were shown to be protected against peroxidation of lipids as stimulated by metal ions with enzymic or non enzymic reactants. Furthermore feeding with tea extracts in normal as well as triton WR-1339 induced hyperlipidemic rats caused decrease in their plasma levels of total cholesterol, phospholipids and triglycerides. The antioxidant and lipid lowering activities of both extracts from CTC leaf and dust tea was comparable and may be due to the presence of natural products like catechin and others.

Decreased reactive oxygen generation during H2O2 decomposition in the presence of samples from human rectal cancer.

Reactive oxygen species (ROS) have generated a great deal of interest in the clinical field since experimental studies showed the involvement of these species in carcinogenesis. This paper reports the detection of ROS during the decomposition of H2O2 in the presence of samples obtained from tissues of 16 patients with rectal carcinoma (age 64 +/- 9 years) operated on in the Division of Surgical Oncology of Pomeranian Medical University, Szczecin (Poland). The samples were cut from the middle of the resected tumors and from the colonic mucosa (10 cm distant from the tumor and free of disease); they were processed and the supernatants, representing the soluble fraction, were used for measurements. Various methods for measuring free radical activity of the examined samples were used, such as chemiluminescence, fluorescent probe 2',7'-dichlorodihydrofluorescein, spin trap 5,5-dimethyl-pyrroline-1-oxide and EPR, the spectrophotometrically examined formation of diformazan during reduction of the p-nitroblue tetrazolium salt, and bleaching of p-nitrosodimethylalanine. A statistically significant difference (P < 0.001) was noticed in mean chemiluminescence +/- standard error of the mean in the presence of the tumor samples (42.6 +/- 7.3) in comparison to the control samples (234.6 +/- 36.0). Significantly decreased generation of ROS from the decomposition of H2O2 in the presence of the tumor samples in comparison to the control samples was also observed when the above-mentioned methods were used. Tumor samples had significantly lower superoxide dismutase activity (33 +/- 4 U/mg protein) than controls (93 +/- 14 U/mg, P < 0.001), which should contribute to a lower capacity of endogenous H2O2 production and therefore less ROS generation upon H2O2 decomposition. We conclude that the tested samples have different redox properties; this supports a possible role of ROS activity during carcinogenesis. Moreover, we propose a new, simple, and sensitive chemiluminescent method, which might be effective in sample differentiation.

Effect of Trophic Status on the Culturability and Activity of Bacteria from a Range of Lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.

Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations.

Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Comparative histochemical and immunohistochemical study on xanthine oxidoreductase/xanthine oxidase in mammalian corneal epithelium

We have previously found that xanthine oxidase (one form of xanthine oxidoreductase that generates reactive oxygen species, such as superoxide radicals and hydrogen peroxide) is present in corneal epithelium of normal rabbit eye. It was suggested that the reactive oxygen species contribute to additional eye damage related to prolonged continuous contact lens wear and irradiation of the eye with UV-B light. To further explore the potential danger of xanthine oxidase as a source of reactive oxygen species, we have examined in the present paper whether xanthine oxidoreductase and xanthine oxidase are present in corneal epithelium of other mammalian species, employing immunohistochemical and enzyme histochemical methods. In corneal epithelium of normal eyes of ox, pig, guinea-pig, and rat xanthine oxidoreductase activity was detected by the tetrazolium salt reduction method and xanthine oxidase activity was localized by a method based on cerium ions capturing hydrogen peroxide. For the immunohistochemical demonstration of the enzymes, rabbit anti-bovine xanthine oxidase antibody, rabbit anti-human xanthine oxidase antibody and monoclonal mouse anti-human xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody were used. The immunohistochemical and enzyme histochemical results show that xanthine oxidoreductase and xanthine oxidase are present both as proteins and as active enzymes in the corneal epithelium of all animals studied. It is hypothesized that under various pathological states, xanthine oxidase-generated reactive oxygen species might contribute to eye damage.

Circadian structure of rat neutrophil phagocytosis

Published findings regarding the time structure of phagocytosis appear to be partly discordant. In addition, this feature has not yet been evaluated in rats, although the rat is an important biomodel used for haematological preclinical biomedical studies. Thus, we examined selected characteristics using rats in order to help elucidate the above-mentioned controversies and to provide further complete data on the haematology of this biomodel. The ingestion of foreign particles (HEMA) or large cells (yeast), the reduction of nitroblue tetrazolium salts (NBT) in rat circulating neutrophils and their migration were evaluated. We found circadian variations in the following characteristics of neutrophil phagocytosis: (i) phagocytic activity; the concentration of engulfed particles and phagocytic index decreases late in the day and peaks in the morning; (ii) NBT reduction; a rise being observed at noon and a fall in the evening. The acrophase for phagocytosis of larger yeast cells was earlier (small hours) than that of smaller HEMA particles (in the morning). Chemotactically oriented migration showed a significant increase in the afternoon, but we have not found a statistically significant fit for the cosine function of this characteristic. No circadian rhythm was present in spontaneous migration. Our findings support the opinion that changes in phagocytic characteristics are a part of the circadian system of the immune system in laboratory rats. By comparing our data with the literature it seems that discrepancies in the courses of the circadian rhythms can be at least partly caused by different laboratory procedures as well as by different acrophases for the various elements of phagocytosis.

Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.

Analysis of elicitor-induced cell viability changes in Lycopersicon esculentum Mill. suspension culture by different methods

Viability changes and hydrogen peroxide production in cultured Lycopersicon esculentum cv. Rio Grande (tomato) cells exposed to induced stress conditions have been investigated. Our data suggest that in order to obtain reliable and insightful data when the hypersensitive response is being evaluated as an early elicitor-induced viability change in suspension cultures, it is strongly advisable to use the assay based on the reduction of tetrazolium salts, since it provides an adequate indicator of changes in metabolic activity that could otherwise be overlooked.

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome p450 induction studies.

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide.

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.

Appraisal of the MTT-based Assay as a Useful Tool for Predicting Drug Chemosensitivity in Leukemia

The MTT-based assay relies upon the cellular reduction of tetrazolium salts to their intensely colored formazans. The test is easy to perform in hematological malignancies and is adaptable for high throughput of samples, although there are some minor limitations in its application resulting from metabolic interference. This class of assays are highly accurate for predicting drug resistance, whereas their predictive value for drug sensitivity depends on the type of disease and drug or drug combination used. They have been found to predict clinical response to fludarabine FLD in B-CLL and were useful for predetermining clinical potential of a single drug or drug combination in AML patients. Extensive studies with ALL patients have supported their advantage for selecting effective drug treatment of the disease. To conclude, pretreatment chemosensitivity assays may help in the selection of chemotherapeutic drugs with the greatest likelihood for clinical effectiveness, and in the exclusion of uneffective therapy. This can lead to improved disease management, response, survival and use of financial resources.