Differential pulse polarographic determination of plasma menadione

A differential pulse polarographic assay for plasma vitamin K₃ (menadione) has been developed. Details of the assay are (i) lipid-soluble material is extracted from plasma into ether by the method of Bjornsson et al. [(1978) Thromb. Haemostas.2, 466–473]; (ii) ether is evaporated under nitrogen and the residue is dissolved in the supporting electrolyte, methanol: 0.2 m borate buffer (9:1), pH 6.8; (iii) current height is measured at ?0.32 V vs SCE on the differential pulse polarogram. The lower sensitivity limit of this technique after addition of standard vitamin K₃ to plasma is 0.3 μm; the calibration curve is linear from 0.6 through 10 μm. Two patients treated with a single dose of menadiol sodium diphosphate, 20 mg/M² i.m., achieved measurable plasma vitamin K₃ levels at 0.5 to 1.0 h ranging between 0.5 (0.08 μg/ml) and 2 μm (0.3 μg/ml).     

Effects of various buffers, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and NaN3 on menadione mediated photophosphorylation in isolated chloroplasts

The effects of DCMU and NaN₃ were studied on menadione-mediated photophosphorylation in broken spinach chloroplasts kept in low oxygen tension in Tricine or HEPES buffers at either high or reduced irradiances. – (A) At high irradiance (131 W. m^?2) and absence of NaN₃ the ATP formation was inhibited by DCMU regardless of the type of buffer used. – (B) At high irradiance and presence of NaN₃ some concentrations of DCMU stimulated, whilst others inhibited the ATP formation in a HEPES buffer. The ATP formation was predominantly inhibited by DCMU in a Tricine buffer. – (C) At reduced irradiance (57 W. m^?2) the chloroplasts in a HEPES buffer were almost insensitive towards DCMU both in the presence and absence of NaN₃. – (D) Chloroplasts in a Tricine buffer were slightly stimulated in their ATP formation by DCMU at reduced irradiance either with or without the presence of NaN₃ in the experimental medium. When menadione acts as a terminal electron acceptor, oxygen is consumed on its reoxidation. The results indicate that this process may occur with oxygen released by the splitting of water as the main oxidant. – The data also demonstrate the importance of caution when selecting buffering substances as well as when choosing light intensities for experiments on photophosphorylation in chloroplasts.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

Binding of Polysaccharide to Peptidoglycan in Cell Wall of Streptomyces roseochromogenes

The polysaccharide-peptidoglycan complex, which was prepared with lysozyme from Streptomyces roseochromogenes IAM53 cell walls, was hydrolyzed with lytic enzyme of Flavo-bacterium to separate polysaccharide. The enzymatically prepared polysaccharide (100 mg) contained 500 μmoles of hexoses, 40 μmoles of hexosamines and 31 μmoles of phosphate. Hexoses consisted of mannose and galactose in a molar ratio of 5 to 1. Hexosamines consisted of equimolar glucosamine and muramic acid, a half of which was identified as muramic acid 6-phosphate. The reducing end of the polysaccharide was muramic acid. The polysaccharide extracted with trichloroacetic acid contained no muramic acid-phosphate. So the polysaccharide moiety of S. roseochromogenes cell walls must be linked covalently to 6-position of muramic acid in peptidoglycan through phosphate,     

Differential antioxidant response between two Symbiodinium species from contrasting environments

High sea surface temperature accompanied by high levels of solar irradiance is responsible for the disruption of the symbiosis between cnidarians and their symbiotic dinoflagellates from the genus Symbiodinium. This phenomenon, known as coral bleaching, is one of the major threats affecting coral reefs around the world. Because an important molecular trigger to bleaching appears related to the production of reactive oxygen species (ROS), it is critical to understand the function of the antioxidant network of Symbiodinium species. In this study we investigated the response of two Symbiodinium species, from contrasting environments, to a chemically induced oxidative stress. ROS produced during this oxidative burst reduced photosynthesis by 30 to 50% and significantly decreased the activity of superoxide dismutase. Lipid peroxidation levels and carotenoid concentrations, especially diatoxanthin, confirm that these molecules act as antioxidants and contribute to the stabilization of membrane lipids. The comparative analysis between the two Symbiodinium species allowed us to highlight that Symbiodinium sp. clade A temperate was more tolerant to oxidative stress than the tropical S. kawagutii clade F. These differences are very likely a consequence of adaptation to their natural environment, with the temperate species experiencing conditions of temperature and irradiance much more variable and extreme.     

Menadione-catalyzed luminol chemiluminescent assay for viability of Mycobacterium bovis

Stable luminol chemiluminescence was observed 10 min after the addition of menadione to a suspension of Mycobacterium bovis homogenized in Middlebrook 7H9 broth base including OADC enrichment. The chemiluminescence intensity was proportional to the absorbance of the bacterial suspension at 600 nm in a range of 0.005 to 0.15. Luminol chemiluminescence disappeared after 10 min incubation of M. bovis at over 60% of ethanol or 4 days of cultivation of M. bovis in the presence of 40 microg/ml of streptomycin. The bacterium showing the disappearance of chemiluminescence could not grow after being washed, suggesting that the inhibition concentration of the antimicrobials can be estimated on the basis of the disappearance of chemiluminescence. Menadione-catalyzed luminol chemiluminescent assay was rapid and sensitive in comparison to turbidimetry, tetrazolium (WST-8) reduction assay, and the assay using the Mycobacteria growth indicator tube (MGIT).     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of amines and neurotransmitters and inhibitors of MAO are useful as neuroprotectants. This work evaluates the human MAO-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, to the directly-acting neurotoxic metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) measured by High-Performance Liquid Chromatography (HPLC), and this approach is subsequently used as a new method for screening of MAO inhibitors and protective agents. Oxidation of MPTP by human MAO-B was more efficient than by MAO-A. R-Deprenyl, a known neuroprotectant, norharman (β-carboline), 5-nitroindazole and menadione (vitamin K3) inhibited MAO-B and reduced the formation of toxic pyridinium cations. Clorgyline and the β-carbolines, harman and norharman, inhibited the oxidation of MPTP by MAO-A. Cigarette smoke, as well as the naturally occurring β-carbolines (norharman and harman) isolated from smoke and coffee inhibited the oxidation of MPTP by MAO-B and/or MAO-A, suggesting protective effects against MPTP. The results show the suitability of the approach used to search for new MAO inhibitors with eventual neuroprotective activity.     

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

Menadione-catalyzed H₂O₂ production by viable cells was proportional to viable cell number, and the assay of this H₂O₂ production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD₅₀ test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H₂O₂ production assay. IC₅₀ of each substance required for 50% inhibition of menadione-catalyzed H₂O₂ production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD₅₀ and IC₅₀, respectively. These results show that menadione-catalyzed H₂O₂ production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification of Photosystem II particles from Phormidium laminosum using the detergent dodecyl-β-d-maltoside. Properties of the purified complex

The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O₂/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (M_r 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.