Optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma

The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid–liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 °C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ = 4.078 ng/ml for tramadol, respectively LLOQ = 3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV% = 5.147% and bias% = − 7.273% in the intra-days and CV% = 4.894% and bias% = 0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV% = 11.517% and bias% = 0.337% in the intra-days and CV% = 6.41% and bias% = 3.259% in the between-days assay.

In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at − 20 °C, but also for 48 h at 15 °C in the re-eluted solution after liquid–liquid extraction.     

Enzyme-linked immunosorbent assay for 4-hydroxynonenal-histidine conjugates

Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE-protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE-histidine (HNE-His) adducts. The method was calibrated using HNE-albumin conjugates as standards (R² = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE-His/mg of protein), precision ( ± 8% intra-assay and ± 12% inter-assay) and spiking recovery ( ± 9%). The assay revealed 60-fold increase of cellular HNE-His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE-His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.     

Ca-activation and stretch-activation in insect flight muscle

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].     

Effects of burial and erosion on the seagrass Zostera noltii

The effects of experimental burial and erosion on the seagrass Zostera noltii were assessed through in situ manipulation of the sediment level (− 2 cm, 0 cm, + 2 cm, + 4 cm, + 8 cm and + 16 cm). Shoot density, leaf and sheath length, internode length, C and N content and carbohydrates of leaves and rhizomes were examined 1, 2, 4 and 8 weeks after disturbance. Both burial and erosion resulted in the decrease of shoot density for all the sediment levels. The threshold for total shoot loss was between 4 cm and 8 cm of burial, particularly during the 2nd week. A laboratory experiment confirmed that shoots did not survive more than 2 weeks under complete burial. There was no evidence of induced flowering by burial or erosion. As well, no clear evidence was found of sediment level effects on leaf and sheath length. Longer rhizome internodes were observed as a response to both burial and erosion, suggesting a plant attempt to relocate the leaf-producing meristems closer to sediment surface or in search of new sediment avoiding the eroded area. The C content of leaves and rhizomes, as well as the non-structural carbohydrates (mainly the starch in rhizomes), decreased significantly along the experimental period, indicating the internal mobilization of carbon to meet the plant demands as a consequence of light deprivation. The significant decrease of N content in leaves, and its simultaneous increase in rhizomes, suggests the internal translocation of nitrogen from leaves to rhizomes. About 50% of the N lost by the leaves was recovered by the rhizomes. Our results indicated that Z. noltii has a high sensitivity to burial and erosion disturbance, which should be considered in the management of coastal activities.     

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sj?berg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Late Holocene lake-level fluctuations in Walker Lake, Nevada, USA

Walker Lake, a hydrologically closed, saline, and alkaline lake, is situated along the western margin of the Great Basin in Nevada of the western United States. Analyses of the magnetic susceptibility (χ), total inorganic carbon (TIC), and oxygen isotopic composition (δ¹⁸O) of carbonate sediments including ostracode shells (Limnocythere ceriotuberosa) from Walker Lake allow us to extend the sediment record of lake-level fluctuations back to 2700 years B.P. There are approximately five major stages over the course of the late Holocene hydrologic evolution in Walker Lake: an early lowstand (> 2400 years B.P.), a lake-filling period ( 2400 to  1000 years B.P.), a lake-level lowering period during the Medieval Warm Period (MWP) ( 1000 to  600 years B.P.), a relatively wet period ( 600 to  100 years B.P.), and the anthropogenically induced lake-level lowering period (< 100 years B.P.). The most pronounced lowstand of Walker Lake occurred at  2400 years B.P., as indicated by the relatively high values of δ¹⁸O. This is generally in agreement with the previous lower resolution paleoclimate results from Walker Lake, but contrasts with the sediment records from adjacent Pyramid Lake and Siesta Lake. The pronounced lowstand suggests that the Walker River that fills Walker Lake may have partially diverted into the Carson Sink through the Adrian paleochannel between 2700 to 1400 years B.P.     

Sleep biological rhythms in normal infants and those at high risk for SIDS

The focus of this study was on daytime and nighttime sleep and wakefulness during the peak age for Sudden Infant Death Syndrome (SIDS), two to four months, to determine whether there are differences between at-risk for SIDS (R) and control (C) infants. Such differences may provide insight on the frequent occurrence of SIDS in the early morning hours, when most babies are asleep. This is the only study in which R and C infants were continuously monitored for long periods of time (24-48 h) and then followed and recorded at monthly intervals until the age of 4-6 months. Data analyses indicate that ultradian REM/NREM cyclicity becomes stabilized into a regular pattern at three months of age. Infants at this age convert from a polyphasic sleep/wakefulness pattern to a circadian one. Among the changes that occur is a lengthening of short sleep periods that consolidate at night and wake periods that consolidate in the daytime. The most striking effects are related to sleep state and vary according to age and sex. The lengthening of single sleep and wakeful periods is coupled with the maturation of the brain. The development of the central nervous system facilitates the synchronization of sleeping patterns with external light input and social entrainment. One or more biological clocks or oscillators may be responsible for these REM/NREM patterns and circadian cycles. These differences during the early morning hours, when the occurrence of SIDS peaks, may have important implications for understanding the pathophysiological mechanism of SIDS.     

Pharmacological effects of vinorelbine on body temperature and locomotor activity circadian rhythms in mice

The effects of vinorelbine (VRL) on the circadian rhythms in body temperature and locomotor activity were investigated in unrestrained B6D2F₁ mice implanted with radio-telemetry transmitters. A single intravenous VRL dose (24 or 12 mg/kg) was given at 7 h after light onset (HALO), a time of high VRL toxicity, and resulted in transient suppression of temperature and activity circadian rhythms in mice kept in light-dark (LD) 12h:12h. Such suppression was dose-dependent. It occurred within 1-5 d after VRL dosing. Recovery of both rhythms was partially complete within 5 d following the high dose and within 2 or 3 d after the low dose and was not influenced by suppression of photoperiodic synchronization by housing in continuous darkness. Moreover, VRL induced a dose-dependent relative decrease in amplitude and phase shift of the temperature circadian rhythm. The mesor and amplitude of the activity rhythm were markedly reduced following the VRL administration. The relevance of VRL dosing time was studied in mice housed in LD 12h:12h. Vinorelbine was injected weekly (20 mg/kg/injection) for 3 wk at 6 or 18 HALO. Vinorelbine treatment ablated the rest-activity and temperature rhythms 3-6 d after each dose, with fewer alterations after VRL dosing at 18 HALO compared to 6 HALO, especially for the body temperature rhythm. There was at least partial recovery 1 wk after dosing, suggesting the weekly schedule of drug treatment is acceptable for therapeutic purposes. Our findings demonstrate that VRL can transiently, yet profoundly, alter circadian clock function. Vinorelbine-induced circadian dysfunction may contribute to the toxicokinetics of this and possibly other anticancer drugs.     

Properties of Pseudomonas AM1 primary-amine dehydrogenase immobilized on agarose.

1. The primary-amine dehydrogenase of Pseudomonas AM1 (primary amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.-) was purified by an improved method and covalently attached to cyanogen bromide-activated Sepharose 4B. The immobilized enzyme showed very little change in its sensitivity to heat and to inhibition by semicarbazide as compared with the soluble enzyme, but had enhanced stability at 0 degrees C. The pH optimum of the immobilized enzyme remained unchanged at pH 7.4. 2. A new type of spectrophotometric assay is described in which sedimentation of the immobilized enzyme in the cuvette is prevented by increasing the viscosity by the presence of 10% (w/w) polyethylene glycol (M1 20 000). Detailed kinetic analysis using this assay showed only insignificant differences in the Km values for n-butylamine and phenazine methosulphate between the soluble and Agarose-bound enzymes. The results are compared with those for other oxidoreductase enzymes immobilized on Sepharose.     

Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.     

Specific interactions between rotavirus outer capsid proteins VP4 and VP7 determine expression of a cross-reactive, neutralizing VP4-specific epitope.

We previously reported that the expression of rotavirus phenotypes by reassortants was affected by recipient genetic background and proposed specific interactions between the outer capsid proteins VP4 and VP7 as the basis for the phenotypic effects (D. Chen, J. W. Burns, M. K. Estes, and R. F. Ramig, Proc. Natl. Acad. Sci. USA 86:3743-3747, 1989). A neutralizing, cross-reactive VP4-specific monoclonal antibody (MAb), 2G4, was used to probe the protein-protein interactions. The VP4 specificity of 2G4 was confirmed by immunoblot analysis. MAb 2G4 reacted with both standard (SA11-C13) and variant rotavirus SA11 (SA11-4F) but did not react with bovine rotavirus B223 as determined by plaque reduction neutralization (PRN) and enzyme-linked immunosorbent assay (ELISA). When a panel of SA11-4F/B223 and SA11-Cl3/B223 reassortants in purified or crude lysate form that had been grown in the presence or absence of trypsin was analyzed with MAb 2G4 by PRN and ELISA, the results with some reassortants were unexpected. That is, MAb 2G4 reacted with VP4 of SA11 parental origin (4F or C13) when it was assembled into capsids with the homologous SA11 VP7 but failed to react with VP4 of SA11 assembled into capsids with heterologous B223 VP7. Conversely, MAb 2G4 failed to react with VP4 of B223 parental origin when it was assembled into capsids with homologous B223 VP7 but did react with B223 VP4 assembled into capsids with the heterologous SA11 VP7. Similar reactivity was observed when 2G4 was used to immunoprecipitate purified double-shelled virions. When soluble unassembled viral proteins were analyzed by ELISA, the 2G4 reactive pattern was as predicted from the parental origin of VP4. That is, 2G4 reacted with the soluble VP4 of reassortants having VP4 from SA11-Cl3 or SA11-4F and failed to react with VP4 of B223 origin, regardless of the origin of VP7. PRN and ELISA results obtained with nonglycosylated viruses revealed that the unexpected reactivity of 2G4 with virus particles was not the result of differential glycosylation of VP7 and epitope masking. These results indicate that the 2G4 epitope existed in the soluble form of VP4 encoded by SA11-Cl3 or SA11-4F but not in soluble B223 VP4. On the other hand, in assembled virions, the presentation of the 2G4 epitope on VP4 was unexpected in some reassortants and was affected by the specific interactions between VP4 and VP7 of heterologous parental origin.     

A rapid and non leaky way for preparation of the sharp intracellular recording microelectrodes

To fill microelectrodes using backfilling method needs excessive time approximately 4–6  h. It is often difficult to fill microelectrodes without damage or leakage. A main problem is bubble formation in microelectrodes which has an impact on the electrical properties of the electrode and thus it influences the quality of the recording. Based on Archimede's principle there is a force within a solution which pushes insoluble material with a lower specific gravity upward and outside of the solution. Centrifugation can increase the force to eliminate the bubbles.

We designed a microelectrode holder to protect microelectrode sensitive tips from mechanical damage due to the gravity tensions; it can help to eliminate the bubbles easily and simultaneously in 10  min or less.

The tests were performed for 2000, 4000, and 8000  rpm centrifugation each one for 3, 6 and 12  min duration respectively, it was found that the bubbles were completely eliminated at 8000  rpm for 6–12  min and there were no significant differences for resistance, and the number of leaky or damaged electrodes between the two methods.

In the new design of devices, the materials used and the design of the holder are simple and the approach is applicable to many laboratories worldwide.     

2,3-BUTANEDIONE INACTIVATES THE [H]NITRENDIPINE BINDING SITES, WHEREAS DIETHYLPYROCARBONATE DOES NOT

The modification of [³H]nitrendipine binding sites in rabbit brain membranes with 2,3-butanedione and diethylpyrocarbonate was investigated. 2,3-Butanedione, an arginine-specific reagent, causes a dose- and time-dependent decrease in the number of [³H]nitrendipine binding sites without altering its dissociation constant. Scatchard analysis of the binding data shows that 50 mM 2,3-butanedione decreases the binding capacity of [³H]nitrendipine from a control value of 71 ± 6 fmol/mg of protein to 40 ± 3 fmol/mg of protein. Complete and selective protection against inactivation is provided by nifedipine. No decrease of [³H]nitrendipine binding occurs when membranes are pretreated with selective histidine reagent diethylpyrocarbonate. The results indicate that arginine but not histidine residue in

Full-size image

-type calcium channel domain is critical for [³H]nitrendipine binding. Copyright © 1996 Elsevier Science Ltd     

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) in human plasma. Acidified plasma is deproteinized with acetonitrile which is separated from the aqueous layer by adding sodium chloride. ASA and SA are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: Novapak C₁₈ 4 μm silica,150×4mm I.D.; eluent: 740 ml water, 900 μl 85% orthophosphoric acid, 180 ml acetonitrile) and photometric detection (237 nm). 2-Methylbenzoic acid is used as internal standard. The method allows the determination of ASA and SA in human plasma as low as 100 ng/ml with good precision (better than 10%). The assay was used to determine the pharmacokinetic parameters of ASA and SA following oral administration of 100–500 mg ASA in healthy volunteers.     

Protection against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide: the role of iron chelation

The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H₂O₂-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H₂O₂ in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C₂, C₃ double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe²⁺]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman's correlation coefficient, ρ = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H₂O₂-induced DNA damage.     

A new ultraviolet-light sensitive mutant of Neurospora crassa with unusual photoreactivation property

A mutant, uvs-(SA3B), which shows high sensitivity to UV light segregated among the progeny in a back-cross of a presumptive MMS-sensitive mutant to a wild-type strain. At 37% survival, this mutant was approximately 5 times more sensitive to UV and also 6 times more sensitive to 4-NQO than the wild type. But it was only slightly sensitive to gamma-ray, MMS, MNNG, MTC and histidine. It showed an unusual photoreactivation response. Its time course of photorecovery was similar to the photoreactivation-defective strain upr-1 of Neurospora crassa. Mutation induction by UV at the ad-3 loci in this mutant strain was lower than that at the same loci in the wild-type strain. The uvs-(SA3B) mutant maps between met-1 and col-4 in linkage group IV, and it was not allelic with the mutagen-sensitive mutant mus-8 which is located in this area. We have concluded, therefore, that uvs-(SA3B) has resulted from mutation in a new DNA-repair gene. This new mutant was barren in homozygous crosses.