vitamin-k-semiquinone-radical and menadiol

Based on the activity of menadione (M) in the human tumor stem cell assay, we conducted a phase I trial of M in patients with advanced cancer. Forty patients (19 men, 21 women) were treated with 90 courses of M; 82 treatment courses are evaluable for toxicity. The median patient age, Karnofsky performance status, and number of prior chemotherapy regimens were 61 years (range 32-74 years), 80% (range 50-100%), and two, respectively. M was given by a short (1-5 h) intravenous infusion every 3 weeks, starting at 40 mg/m2 and escalating by modified Fibonacci scheme to 1360 mg/m2. Toxicity was graded according to the Southwest Oncology Group toxicity scale with defined hypersensitivity reaction (HSR) scales. No grade > or =2 hematologic toxicity was observed. Non-hematologic toxicity consisted of a HSR syndrome of paresthesiae of the extremities, facial flushing, burning of the eyes and mucous membranes, chest pain and dyspnea. HSR was defined as Grade I toxicity by the presence of facial numbness, flushing, and/or a tingling sensation or burning of the eyes and mucous membranes. Grade II toxicity was defined as the presence of the same above symptoms plus chest tightness, paresthesiae of extremities and/or dyspnea and chest pain. These toxicities were grade 1 in 3 of 4 patients at a dose of 840 mg/m2. At 1360 mg/m2, 2 of 13 patients suffered grade 1 HSR and 7 of 13 grade 2 HSR. No objective partial or complete responses were observed. Plasma menadione concentrations peaked at 1.9-7.4 microM during the infusion in 3 patients receiving 1360 mg/m2. Further phase 1 and 2 combination trials using longer infusion durations have resulted from this trial.

Topics: Adult; Aged; Antineoplastic Agents; Dose-Response Relationship, Drug; Female; Humans; Infusions, Intravenous; Karnofsky Performance Status; Male; Middle Aged; Neoplasms; Vitamin K

To evaluate the efficacy of oral menadiol compared to intravenous phytomenadione when correcting coagulopathies associated with cholestasis.. A total of 26 patients with cholestasis and an international normalized ratio (prothrombin time) greater than 1.2, were randomized to receive either 20 mg o.d. for 3 days of oral menadiol (n=12), or 10 mg o.d. of intravenous phytomenadione (n=14) prior to endoscopic retrograde cholangeopancreatography. Liver function tests and international normalized ratio were measured daily for 3 days.. Liver function tests and international normalized ratio were comparable between groups at entry into the study (P > 0.05), but serum albumin was significantly lower in the intravenous phytomenadione group following treatment (P < 0.05). A decrease in international normalized ratio occurred in both groups following administration of vitamin K (P < 0.05). Two patients in the intravenous group required fresh frozen plasma, as failure to normalize international normalized ratio was observed. No adverse drug reactions were observed in either group, and no patient required re-admission for bleeding during a 4-week follow-up period after cholangeopancreatography.. Oral menadiol appears to be an effective alternative to intravenous phytomenadione in the correction of coagulopathies associated with obstructive liver disease. This simplifies the care of patients with deranged clotting times requiring cholangeopancreatography, particularly those to be managed as out-patients.

Topics: Aged; Aged, 80 and over; Blood Coagulation Disorders; Cholestasis; Female; Humans; Male; Middle Aged; Prothrombin Time; Vitamin K; Vitamin K 1

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme providing cytoprotection from quinone species. In addition, it is expressed at high levels in many human tumors, such as breast cancer. Therefore, it is considered to be a potential target in cancer treatment. In order to detect intracellular NQO1 activity in MCF-7 aggregates as a cancer model, we present, in this study, a double-mediator system combined with large-scale integration (LSI)-based amperometric devices. This LSI device contained 20 × 20 Pt working electrodes with a 250 μm pitch for electrochemical imaging. In the detection system, menadione (MD) and [Fe(CN)

Topics: Cell Respiration; Electrochemical Techniques; Electron Transport Complex I; Enzyme Assays; Ferricyanides; Humans; MCF-7 Cells; NAD(P)H Dehydrogenase (Quinone); Optical Imaging; Oxidation-Reduction; Rotenone; Vitamin K; Vitamin K 3

The endogenous neuropeptide galanin is ubiquitously expressed throughout the mammalian brain. Through the galanin receptors GalR1-3, galanin has been demonstrated to modulate both glutamatergic and GABAergic neurotransmission, and this appears to be important in epilepsy and seizure activity. Accordingly, galanin analogues are likely to provide a new approach to seizure management. However, since peptides are generally poor candidates for therapeutic agents due to their poor metabolic stability and low brain bioavailability, a search for alternative strategies for the development of galanin-based anti-convulsant drugs was prompted. Based on this, a rationally designed GalR1 preferring galanin analogue, NAX-5055, was synthesized. This compound demonstrates anti-convulsant actions in several animal models of epilepsy. However, the alterations at the cellular level leading to this anti-convulsant action of NAX-5055 are not known. Here we investigate the action of NAX-5055 at the cellular level by determining its effects on excitatory and inhibitory neurotransmission, i.e. vesicular release of glutamate and GABA, respectively, in cerebellar, neocortical and hippocampal preparations. In addition, its effects on cell viability and neurotransmitter transporter capacity were examined to evaluate potential cell toxicity mediated by NAX-5055. It was found that vesicular release of glutamate was reduced concentration-dependently by NAX-5055 in the range from 0.1 to 1000 nM. Moreover, exposure to 1 μM NAX-5055 led to a reduction in the extracellular level of glutamate and an elevation of the extracellular level of GABA. Altogether these findings may at least partly explain the anti-convulsant effect of NAX-5055 observed in vivo.

Topics: Animals; Animals, Newborn; Anticonvulsants; Brain; Cell Survival; Cells, Cultured; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Female; Galanin; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lipopeptides; Male; Mice; Mice, Inbred C57BL; N-Methylaspartate; Neurons; Neurotransmitter Agents; Organ Culture Techniques; Pregnancy; Time Factors; Vitamin K

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

Topics: Bacterial Proteins; Cysteine; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Mutation; Protein Kinases; Pseudomonas putida; Vitamin K

Topics: Aged, 80 and over; Antifibrinolytic Agents; Humans; Male; Vitamin K; Vitamin K Deficiency Bleeding

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc1 complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa3 oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa3 oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.

Topics: Cytochrome c Group; Cytochromes a; Cytochromes a3; Electron Transport Complex III; Fluorescence; Multigene Family; Rhodothermus; Vitamin K

We have studied the transient kinetics of quinol-dependent heme reduction in Escherichia coli nitrate reductase A (NarGHI) by the menaquinol analogue menadiol using the stopped-flow method. Four kinetic phases are observed in the reduction of the hemes. A transient species, likely to be associated with a semiquinone radical anion, is observed with kinetics that correlates with one of the phases. The decay of the transient species and the formation of the second reduction phase of the hemes can be fitted to a double-exponential equation giving similar rate constants, k(1) = 9.24 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the decay of the transient species, and k(1) = 9.23 +/- 0.9 s(-1) and k(2) = 0.22 +/- 0.02 s(-1) for the formation of the reduction phase. The quinol-binding-site inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and stigmatellin have significant and different inhibitory effects on the reduction kinetics. The kinetics of heme reduction in NarI expressed in the absence of the NarGH catalytic dimer (NarI(DeltaGH) exhibits only two kinetic phases, and the decay of the transient species also correlates kinetically with the second reduction phase of the hemes. We have also studied nitrate-dependent heme reoxidation following quinol-dependent heme reduction using a sequential stopped-flow method. HOQNO elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes. On the basis of our results, we propose schemes for the mechanism of NarGHI reduction by menaquinol and reoxidation by nitrate.

Topics: Catalysis; Dimerization; Enzyme Inhibitors; Escherichia coli; Heme; Hydroxyquinolines; Kinetics; Models, Chemical; Mutagenesis, Site-Directed; Nitrate Reductases; Nitrates; Oxidation-Reduction; Polyenes; Vitamin K

We have studied the effects of site-directed mutations in Escherichia coli nitrate reductase A (NarGHI) on heme reduction by a menaquinol analogue (menadiol) using the stopped-flow method. For NarGHI(H66Y) and NarGHI(H187Y), both lacking heme b(L) but having heme b(H), the heme reduction by menadiol is abolished. For NarGHI(H56R) and NarGHI(H205Y), both without heme b(H) but with heme b(L), a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. These results indicate that electrons from menadiol oxidation are transferred initially to heme b(L). A transient species, likely to be associated with a semiquinone radical anion, was generated not only on reduction of the wild-type enzyme as observed previously (1) but also on reduction of NarGHI(H56R) and NarGHI(H205Y). The inhibitors 2-n-heptyl-4-hydroxyquinoline-N-oxide and stigmatellin both have significant effects on the reduction kinetics of NarGHI(H56R) and NarGHI(H205Y). We have also investigated the reoxidation of menadiol-reduced heme by nitrate in the mutants. Compared to the wild type, no significant heme reoxidation is observed for NarGHI(H56R) and NarGHI(H205Y). This result indicates that a single mutation removing heme b(H) blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH.

Topics: Arginine; Electron Transport; Escherichia coli Proteins; Heme; Histidine; Hydroxyquinolines; Mutagenesis, Site-Directed; Naphthols; Nitrate Reductase; Nitrate Reductases; Nitrates; Oxidation-Reduction; Polyenes; Spectrometry, Fluorescence; Terpenes; Tyrosine; Vitamin K

The nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and also defective in either ubiquinone or menaquinone biosynthesis. The data reveal that Nap is coupled more effectively to menaquinol oxidation than to ubiquinol oxidation. Conversely, parallel experiments with a second set of mutants revealed that nitrate reductase A couples more effectively with ubiquinol than with menaquinol. Three further sets of strains were constructed with combinations of in frame deletions of ubiCA, menBC, napC, napF and napGH genes. NapF, NapG and NapH were shown to play no role in electron transfer from menaquinol to the NapAB complex but, in the Ubi+Men- background, deletion of napF, napGH or napFGH all resulted in total loss of nitrate-dependent growth. Electron transfer from ubiquinol to NapAB was totally dependent upon NapGH, but not on NapF. NapC was essential for electron transfer from both ubiquinol and menaquinol to NapAB. The results clearly established that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi+Men+ strain implicates NapF in an energy- conserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energy- conserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC.

Topics: Energy Metabolism; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Kinetics; Nitrate Reductase; Nitrate Reductases; Nitrates; Operon; Oxidation-Reduction; Protein Subunits; Ubiquinone; Vitamin K

We have found that short chain plastoquinones effectively stimulated photoreduction of the low potential form of cytochrome b(559) and were also active in dark oxidation of this cytochrome under anaerobic conditions in Triton X-100-solubilized photosystem II (PSII) particles. It is also shown that molecular oxygen competes considerably with the prenylquinones in cytochrome b(559) oxidation under aerobic conditions, indicating that both molecular oxygen and plastoquinones could be electron acceptors from cytochrome b(559) in PSII preparations. alpha-Tocopherol quinone was not active in the stimulation of cytochrome photoreduction but efficiently oxidized it in the dark. Both the observed photoreduction and dark oxidation of the cytochrome were not sensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. It was concluded that both quinone-binding sites responsible for the redox changes of cytochrome b(559) are different from either the Q(A) or Q(B) site in PSII and represent new quinone-binding sites in PSII.

Topics: Aerobiosis; Anaerobiosis; Cytochrome b Group; Darkness; Electron Transport; Hydrogen Peroxide; Kinetics; Light; Models, Biological; Octoxynol; Oxidation-Reduction; Oxygen; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Plastoquinone; Solubility; Spectrophotometry; Vitamin E; Vitamin K

It has been suggested that the enzymes DT-diaphorase and superoxide dismutase act in concert to prevent redox cycling of naphthoquinones and thus protect against the toxic effects of such substances. Little is known, however, about the scope of this process or the conditions necessary for its operation. In the presence of low levels of DT-diaphorase, 2-methyl-1,4-naphthoquinone was found to undergo redox cycling. This was very effectively inhibited by SOD, and in the presence of both enzymes the hydroquinone was maintained in the reduced form. The inhibitory effect of the enzyme combination was overcome, however, at high concentrations of the quinone, or by small increases in pH. Furthermore, redox cycling was re-established by addition of haemoproteins such as cytochrome c and methaemoglobin. DT-diaphorase and SOD strongly inhibited redox cycling by 2,3-dimethyl- and 2,3-dimethoxy-1,4-naphthoquinone, but not that of 2-hydroxy-, 5-hydroxy- or 2-amino-1,4-naphthoquinone. Inhibition of redox cycling by a combination of DT-diaphorase and SOD is therefore not applicable to all naphthoquinone derivatives, and when it does occur, it may be overwhelmed at high quinone concentrations, and it may not operate under slightly alkaline conditions or in the presence of tissue components capable of initiating hydroquinone autoxidation.

Topics: Animals; Cattle; Cytochrome c Group; Dicumarol; Electron Transport Complex IV; Hemoglobins; Hydrogen-Ion Concentration; Methemoglobin; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Oxidation-Reduction; Oxygen; Rats; Superoxide Dismutase; Time Factors; Vitamin K

At neutral pH, 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone readily autoxidize to the corresponding quinones. In an unpurified phosphate buffer, the autoxidation of both substances proceeded in a linear fashion after a brief lag phase. Addition of a chelating agent or purification of the buffer decreased the duration of the lag phase of 1,4-naphthohydroquinone autoxidation, but had no effect on the linear rate. In the case of 2-methyl-l,4-naphthohydroquinone, such treatment eliminated the lag phase and greatly increased the linear rate of oxidation. The lag phases and oxidation rates seen in unpurified buffers could be replicated by addition of submicromolar amounts of copper to purified buffer. The effects of low levels of copper were qualitatively similar to those of superoxide dismutase, and it is suggested that the effects of this metal on naphthohydroquinone autoxidation reflects its ability to act as a superoxide dismutase. The relative rates of autoxidation of naphthohydroquinones are important because they may determine the balance between activation and detoxication of naphthoquinones within cells. When measuring such rates, or assessing rates of redox cycling of naphthoquinones, it is important to employ a chelating agent or use highly purified buffers and reagents. Failure to do so may lead to erroneous conclusions concerning the reactivity of naphthohydroquinones and the ability of naphthoquinones to generate "active oxygen" species.

Topics: Animals; Buffers; Cattle; Chelating Agents; Copper; Hydroquinones; In Vitro Techniques; Iron; Kinetics; Oxidation-Reduction; Reactive Oxygen Species; Superoxide Dismutase; Vitamin K

The antioxidant activity of reduced menadione was investigated and compared with that of alpha-tocopherol both in solvent solution and in large unilamellar vesicles by using azocompounds as free radical generators. The results show that: i) reduced menadione behaves as a chain-breaking antioxidant; ii) its inhibition rate constant is similar to that of alpha-tocopherol in homogeneous solution, whereas it is 4 times larger in egg yolk lecithin vesicles; iii) the stoichiometric factor is found lower than 1 in both systems, since a substantial portion of menadiol is consumed by autoxidation and does not contribute to radical trapping; iv) when both alpha-tocopherol and menadiol are present in vesicles, reduced menadione can spare alpha-tocopherol. Data presented here suggest that the reduced form of vitamin K may protect, when present, cellular membranes from free radical damage.

Topics: Antioxidants; Drug Synergism; Liposomes; Oxidation-Reduction; Phosphatidylcholines; Quinones; Solutions; Solvents; Vitamin E; Vitamin K

Inside-out subcellular vesicles of Azotobacter vinelandii are found to produce delta pH and delta psi (interior acidic and positive) when oxidising malate or menadiol. These effects are inherent in both Cyd+ Cyo- (lacking the o-type oxidase) and Cyd- Cyo+ (lacking the bd-type oxidase) strains. They appear to be myxothiazol-sensitive in the Cyd- Cyo+ strain but not in the Cyd+ Cyo- strain. The H+/e- ratio for the terminal part of respiratory chain of a bd-type oxidase overproducing strain is established as being close to 1. It is also shown that NADH oxidation by the vesicles from the Cyd- Cyo+ strain is sensitive to low concentrations of myxothiazol and antimycin A whereas that of the Cyd+ Cyo- strain is resistant to these Q-cycle inhibitors. It is concluded that (i) the bd-type oxidase of A. vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase and (ii) Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A. vinelandii respiratory chain and does not in the bd-type quinol oxidase terminated branch. These relationships are discussed in the context of the respiratory protection function of the bd-type oxidase in A. vinelandii.

Topics: Anaerobiosis; Azotobacter vinelandii; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Fractionation; Cytochrome b Group; Cytochromes; Electron Transport Chain Complex Proteins; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Malates; Methacrylates; Oxidoreductases; Subcellular Fractions; Thiazoles; Valinomycin; Vitamin K

We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered beta subunits. To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified alpha beta gamma complex. With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant. The results as a whole are consistent with a substituted-enzyme mechanism for the reduction of nitrate by the quinols. Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition. Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo. To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with beta subunits that lack an Fe-S centre. The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity. The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity. The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of +80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical.

Topics: Escherichia coli; Hydroquinones; Kinetics; Mutation; Nitrate Reductase; Nitrate Reductases; Vitamin K

The compound known as "synkavit" is a diphosphate derivative of vitamin K3 (menadion), which is capable of being selectively accumulated in certain tumour cells, and covalently bonded to DNA producing considerable DNA damage. On the other hand, iodine-125 nuclide incorporated into the nucleus of living cells causes extreme radiotoxic effects. Consequently, synkavit can be used as a specific carrier of iodine-125 into the nucleus of tumour cells. Thus, its iodo-derivatives have become interesting agents on the potential application of iodine-125 in cancer therapy. 6-Iodo-synkavit is a unique iodo-derivative described in the literature. In addition, its synthesis and radioiodination is still problematic, and consequently the results obtained using 6-iodo-synkavit labelled with iodine-125 remains in question. For this reason, the synthesis of 6-iodo-synkavit was examined in this study. It is finally determined that a mixture of different iodo-isomers of synkavit has been produced rather than its specific 6-iodo-isomer, when the synthetic sequence was begun with the direct sulfonation of 2-methyl-naphthalene. On the other hand, it is also determined that synkavit can directly be radioiodinated using different iodination techniques, and iodogen especially can be successfully used as an oxidative agent.

Topics: Antineoplastic Agents; Iodine Radioisotopes; Isomerism; Isotope Labeling; Urea; Vitamin K

The quinol oxidase appears to be mainly responsible for the oxidation of the bacterial MKH2 in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mumol cytochrome aa3 per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa3 was about 10(3) electrons.s-1 at pH 7 and 37 degrees C. The preparation consisted mainly of a M(r) 57,000 and a M(r) 36,000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M(r) 36,000 polypeptide.

Topics: Amino Acid Sequence; Bacillus subtilis; Chromatography, Ion Exchange; Electron Transport Complex IV; Genes, Bacterial; Hydroquinones; Molecular Sequence Data; Naphthoquinones; Oxidation-Reduction; Vitamin K

The renal processing of the glutathione conjugate of menadione, 2-methyl-3-S-glutathionyl-1,4-naphthoquinone (thiodione) was studied in the isolated perfused rat kidney. Thiodione at an initial concentration of 600 microM was eliminated rapidly from the perfusate (clearance = 6.0 ml/min). Renal disposition could be ascribed to metabolism and transport of the glutathione conjugate. Renal metabolism by gamma-glutamyltranspeptidase was inhibited by AT-125 [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] (0.5 mM) resulting in a reduction of the thiodione clearance to 0.86 ml/min. Further reduction of the renal clearance of thiodione was achieved by a combination of AT-125 (0.5 mM) and probenecid (0.5 mM), resulting in a renal clearance of 0.58 ml/min which equalled glomerular filtration rate. Addition of thiodione to the perfusate caused loss of renal function and cellular damage, as reflected by a decreased glucose reabsorption and an increased urinary secretion of lactate dehydrogenase, respectively. Thiodione-induced nephrotoxicity was ameliorated by AT-125 and prevented completely by a combination of AT-125 and probenecid. Aminooxyacetic acid (0.5 mM), an inhibitor of beta-lyase, did not afford protection against the nephrotoxic action of thiodione. From our results it can be concluded that the thiodione-mediated toxicity in the isolated perfused rat kidney can be linked to cellular uptake by anionic transport systems and metabolism by gamma-glutamyltranspeptidase.

Topics: Animals; Biological Transport; gamma-Glutamyltransferase; Isoxazoles; Kidney; Male; Perfusion; Probenecid; Rats; Rats, Inbred Strains; Vitamin K

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.

Topics: Bacillus; Cholic Acids; Detergents; Electrophoresis, Polyacrylamide Gel; Glucosides; Molecular Weight; Nitrate Reductase; Nitrate Reductases; Sodium Chloride; Vitamin K

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Topics: Alkaline Phosphatase; Animals; Female; Histocytochemistry; Immunohistochemistry; Male; Rats; Rats, Inbred Strains; Staining and Labeling; Substrate Specificity; Vitamin K

A bioassay of vitamin K is described, based on the prothrombin clotting time of 3-week-old, vitamin-K-depleted, and cumatetralyl-sensitized male broiler chicks, using a homologous thrombokinase preparation. With this test it could be shown that the diacetate and dibutyrate esters of menadiol are vitamin-K-active. The bioactivity of menadione from these menadiolesters amounted to about 70% of the standard menadione from a coated menadione sodium bisulfite (Dohyfral). Menadiol seems to be temperature-resistant under such conditions, whereby two uncoated MSB preparations lost about 60% of their activity.

Topics: Animals; Biological Assay; Biological Availability; Chickens; Dogs; Drug Stability; Partial Thromboplastin Time; Vitamin K

Although many synthetic substrates and methods are available for the histochemical detection of non-specific esterases and non-specific acid phosphatase, there are still further possibilities to investigate these hydrolases histochemically. This was shown for menadiol diacetate and menadiol diphosphate using tetrazolium salt, simultaneous azo-dye as well as metal salt methods in many rat tissues. In comparison, the azo-dye procedure with various Fast salts or hexazonium Pararosaniline or New Fuchsin delivered less satisfactory results; precisely localized stain in sufficient amounts was obtained for non-specific esterases using nitro BT, tetranitro BT or benzothiazolystyrylphthalhydrazidyl tetrazolium (BSPT) and for non-specific acid phosphatase with BSPT in the tetrazolium salt method or using cerium ions for phosphate trapping in the diaminobenzidine-nickel-hydrogen peroxide procedure.

Topics: Acid Phosphatase; Animals; Azo Compounds; Esterases; Female; Histocytochemistry; Male; Metals; Rats; Rats, Inbred Strains; Tetrazolium Salts; Vitamin K

Topics: Acidosis, Lactic; Adolescent; Ascorbic Acid; Electron Transport; Energy Metabolism; Female; Humans; Mitochondria, Muscle; Muscular Diseases; Physical Exertion; Vitamin K

The effect of menadiol (vitamin K3) on fresh specimens of human lymphatic neoplasms (HLN) was tested by means of the differential staining cytotoxicity assay. Menadiol was tested alone and in combination with standard antineoplastic agents. Drug effects were then compared with the effects of the same drugs in normal human lymphocytes and in fresh specimens of human non-small cell lung cancer. By itself, menadiol was moderately toxic to HLN, but not to normal lymphocytes or non-small cell lung cancer. Menadiol, menadione, and two structurally related congeners were equitoxic to HLN cells, but sodium metabisulfite (present in menadiol solutions as a preservative) was nontoxic. Menadiol increased the cytotoxic effects of a number of standard agents in HLN but not in normal lymphocytes. Cell survival times with mechlorethamine, vincristine, and dexamethasone were converted from a range characteristic of drug resistance (ie, range observed in relapsed patients) to a range characteristic of drug sensitivity (ie, range observed in untreated patients) in the presence of menadiol. These effects occurred at a concentration (2.0 micrograms/ml; 4.7 microM) of menadiol which is probably clinically achievable and which did not deplete intracellular glutathione. Menadiol should receive clinical testing as a chemosensitizing agent in HLN.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Colony-Forming Units Assay; Dexamethasone; Drug Evaluation, Preclinical; Glutathione; Humans; Leukemia; Lymphoma; Mechlorethamine; Tumor Stem Cell Assay; Vincristine; Vitamin K

Topics: Animals; Dicumarol; Glutathione; Liver; Mice; Microsomes, Liver; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Photochemistry; Quinone Reductases; Rats; Vitamin K

During a Phase I study of intravenous vitamin E-free alcohol (all-rac-alpha-tocopherol or Ephynal) in patients with neuroblastoma, we noticed a bleeding diathesis in two patients receiving 2300 mg/m2 daily for four or more days in succession. Both blood prothrombin time and accelerated partial thromboplastin times were prolonged. These spontaneously returned to normal levels three days after interrupting vitamin E infusions. It was also noted that factors VII, IX and X were decreased, which corresponded with the prolonged PT and APTT. It was found that by infusing menadiol sodium diphosphate just prior to the vitamin E, these inhibiting effects on procoagulant factors could be abrogated and high dosages of vitamin E-free alcohol safely given.

Topics: Blood Coagulation Factors; Child; Child, Preschool; Drug Interactions; Factor IX; Factor VII; Factor X; Humans; Male; Neuroblastoma; Partial Thromboplastin Time; Prothrombin; Prothrombin Time; Vitamin E; Vitamin K

From the results of the alpha-particle track autoradiography, the alkaline phosphatase studies, the biodistribution investigations and the therapeutic experiments with the transplanted Franks and Hemmings (1978) adenocarcinoma of the rectum in mice, an important finding is the striking selectivity of the uptake of the compound 6-211 At-astato-2-methyl-1, 4-naphthoquinol bis (diphosphate salt), after intraperitoneal injection into the tumour cell nuclei and in many cases into the nuclei of tumour stem cells, together with negligible uptake into normal colon and narrow. The compound, 6-211 At-astato-2-methyl-1, 4-naphthoquinol bis (diphosphate salt) has been found to cure about two-thirds of the transplanted adenocarcinoma of the rectum in mice after a single intra-peritoneal injection of 3-4 microCi. This approach can be regarded as a form of metabolically-directed drug targetting on to a tumour product which is an enzyme, an alkaline phosphatase isozyme, present in the cells of some tumours. The compound is being studied further from the point of view of possible human therapeutic applications.

Topics: Alkaline Phosphatase; Animals; Astatine; Bromine; Energy Transfer; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Neoplasms; Phosphorylation; Radioisotopes; Vitamin K

Topics: Antineoplastic Agents; Colonic Neoplasms; Energy Transfer; Humans; Melanoma; Naphthols; Neoplasms; Pancreatic Neoplasms; Skin Neoplasms; Vitamin K

Topics: Animals; Humans; Male; Mice; Mice, Nude; Neoplasms, Experimental; Prostatic Hyperplasia; Prostatic Neoplasms; Tritium; Vitamin K

An initial incubation of bovine thyroid slices with TSH causes decreased responsiveness to the subsequent addition of the hormone when the adenylate cyclase -cAMP system and other metabolic parameters are measured. After the initial incubation with TSH, refractoriness persists despite incubation of thyroid slices for 24 h in the absence of added TSH. Removal of persistently bound TSH by trypsin or antibody to TSH did not reverse the refractoriness during a subsequent 2 h incubation without added TSH. However, normal TSH responsivity was restored by the removal of TSH bound during the first incubation by the addition of either trypsin or antibody to TSH at the beginning of a 24-h second incubation. Restitution of TSH responsiveness after treatment with trypsin or antibody to TSH requires new protein synthesis. While TSH-induced refractoriness does not modify stimulation of cAMP by cholera toxin, its effect on glucose oxidation is significantly diminished. Menadiol stimulation of glucose oxidation is not inhibited in thyroid slices refractory to TSH. Thus, the effect of menadiol is subsequent to the block induced by TSH, whereas that of cholera toxin is proximal to it.

Topics: Adenylyl Cyclases; Animals; Cattle; Cholera Toxin; Cyclic AMP; Glycolysis; In Vitro Techniques; Kinetics; Protein Biosynthesis; Thyroid Gland; Thyrotropin; Vitamin K

Topics: Acid Phosphatase; Animals; Disease Models, Animal; Dogs; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Rats; Rats, Inbred Strains; Substrate Specificity; Vitamin K

The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase.

Topics: Adenosine Monophosphate; Adenylate Kinase; Cell Fractionation; Cyanides; Detergents; Flavin-Adenine Dinucleotide; Mitochondria; Mitosporic Fungi; Nucleotides; Oxidation-Reduction; Oxidoreductases; Oxygen Consumption; Succinates; Vitamin K

This report was prepared at the request of the International Atomic Energy Agency and presented at an Advisory Group Meeting on "Applications of Recent Radiobiological Research in Radiotherapy" held in Vienna, Austria, from 24-28 September 1979. Based on the recommendations of the Advisory Group, the I.A.E.A. has decided to initiate a coordinated international research programme entitled "Exploration of the Possibility of high LET RAdiation for Nonconventional Radiotherapy in Cancers". This programme will explore possible applications of corpuscular radiations (neutrons, protons, accelerated heavy ions, negative pi-mesons, thermal and epithermal neutrons) in cancer therapy. In addition, the programme will include research on in situ radiotherapy with unsealed radionuclides and thermal neutron capture. For further information on the programme, please contact Dr T. IWASAKI, Section of Radiation Biology, Division of Life Sciences, International Atomic Energy Agency, Wagramerstrasse 5, P. O. Box 100, A-1400 Vienna, Austria.

Topics: Animals; Antibodies, Neoplasm; Astatine; Cricetinae; Gallium Radioisotopes; Humans; Idoxuridine; Iodine Radioisotopes; Liposomes; Mice; Neoplasms; Radioisotopes; Tamoxifen; Tritium; Vitamin K

Topics: Amides; Bromine; Chromatography; Dimethylformamide; Diphosphates; Naphthoquinones; Oxidation-Reduction; Phosphates; Phosphorus Isotopes; Phosphorylation; Research; Solvents; Ultraviolet Rays; Vitamin K

Topics: Carcinoma; Carcinoma, Squamous Cell; Cheek; Cobalt Isotopes; Humans; Injections, Intravenous; Mouth Neoplasms; Neoplasms; Tongue Neoplasms; Vitamin K

Topics: Animals; Bilirubin; Hemoglobins; Hemolysis; Hyperbilirubinemia; Jaundice; Rats; Vitamin K

Topics: Bilirubin; Hemostatics; Infant, Newborn; Plasma; Prothrombin; Prothrombin Time; Vitamin K

Topics: Adenosine Triphosphate; Animals; Ascites; Carcinoma; Carcinoma, Ehrlich Tumor; Diphosphates; Mice; Radiation-Sensitizing Agents; Vitamin K

Topics: Bronchi; Bronchial Neoplasms; Carcinoma; Humans; Naphthoquinones; Vitamin K

Topics: Antifibrinolytic Agents; Hexosephosphates; Hexoses; In Vitro Techniques; Monoiodotyrosine; Naphthoquinones; Pentose Phosphate Pathway; Thyroid Gland; Tyrosine; Vitamin K

Topics: HeLa Cells; Humans; Radiation Effects; Radiation Injuries; Tissue Culture Techniques; Vitamin K

Topics: Azotobacter; Azotobacter vinelandii; Inactivation, Metabolic; Vitamin K; Vitamin K 3

Topics: Acetone; Connective Tissue; DNA; Fibroblasts; In Vitro Techniques; Radiation-Protective Agents; Radiation-Sensitizing Agents; Research; Urethane; Vitamin K

Topics: Animals; Ascites; Carcinoma, Ehrlich Tumor; Diphosphates; Mice; Neoplasms, Experimental; Radiation-Sensitizing Agents; Vitamin K

Topics: Antifibrinolytic Agents; Humans; Naphthoquinones; Neoplasms; Retinoids; Vitamin K

Topics: Biochemical Phenomena; Diphosphates; Inactivation, Metabolic; Vitamin K

Topics: Acetates; Cholesterol; Lipid Metabolism; Lipogenesis; Liver; Vitamin K; X-Rays

Topics: Antifibrinolytic Agents; Bilirubin; Humans; Infant, Newborn; Infant, Premature; Naphthoquinones; Vitamin K

Topics: Animals; Cell Respiration; Liver; Phosphates; Rats; Respiration; Vitamin K; X-Rays

Topics: 3-Hydroxyanthranilic Acid; Aminobenzoates; Animals; Antifibrinolytic Agents; Diphosphates; Erythrocytes; Inclusion Bodies; Mice; Sodium; Tryptophan; Vitamin K

Topics: Anemia; Anemia, Hemolytic; Child; Humans; Infant; Infant, Newborn; Infant, Newborn, Diseases; Jaundice; Vitamin K

Topics: Ascorbic Acid; Folic Acid; Niacinamide; Polarography; Riboflavin; Thiamine; Vitamin K; Vitamins

A radiographic method for studying the delay in gastric emptying in the rat caused by whole body irradiation has been applied to the investigation of two compounds which modify the effects of irradiation. The method may be of general use in the study of the action of drugs. Synkavit, which can potentiate the lethal effects of irradiation on tumours, caused a delay in gastric emptying. When Synkavit and irradiation were combined, a simple summation of the delays produced by each of these agents occurred. Cysteamine, which gives some protection against the lethal action of x-rays, also caused a delay in gastric emptying. It did not protect against the action of x-rays on stomach emptying.

Topics: Animals; Antifibrinolytic Agents; Cysteamine; Gastric Emptying; Male; Radiation Effects; Rats; Retinoids; Stomach; Vitamin K

Topics: Erythrocytes; Glycosides; Humans; In Vitro Techniques; Infant; Infant, Newborn; Infant, Premature; Liver; Regeneration; Vitamin K; Vitamins

Topics: 3-Hydroxyanthranilic Acid; Aminobenzoates; Animals; Erythrocytes; Mice; Naphthoquinones; Tryptophan; Vitamin K

Topics: Antifibrinolytic Agents; Humans; Psoriasis; Retinoids; Vitamin K

Topics: Antifibrinolytic Agents; Humans; Infant, Newborn; Infant, Premature; Naphthoquinones; Vitamin K

Topics: Chromosome Aberrations; Chromosome Disorders; Chromosomes; Humans; Vitamin K; X-Rays

Topics: Antifibrinolytic Agents; Hemostatics; Humans; Infant; Infant, Newborn; Naphthoquinones; Prothrombin; Vitamin K; Vitamin K 1

Topics: Antifibrinolytic Agents; Bilirubin; Erythrocytes; Female; Humans; Infant, Newborn; Term Birth; Vitamin K

Topics: Bilirubin; Humans; Infant, Newborn; Vitamin K

Topics: Cysteine; Drug-Related Side Effects and Adverse Reactions; Glutathione; Radiation-Sensitizing Agents; Vitamin K

Topics: Anemia; Anemia, Hemolytic; Child; Humans; Infant; Infant, Premature, Diseases; Vitamin K

Topics: 3-Hydroxyanthranilic Acid; Aminobenzoates; Antifibrinolytic Agents; Carcinogens; Pyridines; Quinolinic Acid; Vitamin K

Topics: 3-Hydroxyanthranilic Acid; Aminobenzoates; Antifibrinolytic Agents; Carcinogens; Pyridines; Quinolinic Acid; Vitamin K

Topics: Escherichia coli; Radiation; Saccharomyces cerevisiae; Vitamin K; X-Rays; Yeasts

Topics: Antifibrinolytic Agents; Hemostatics; Humans; Infant, Newborn; Naphthoquinones; Prothrombin Time; Vitamin K; Vitamin K 1

Topics: Animals; Cell Nucleus; DNA; Fibroblasts; Mice; Nucleic Acids; Tissue Culture Techniques; Vitamin K

Topics: Antifibrinolytic Agents; Hemostatics; Infant, Newborn; Leadership; Prothrombin Time; Vitamin K; Vitamin K 1

Topics: Adenoidectomy; Adenoids; Hemorrhage; Palatine Tonsil; Tonsillectomy; Vitamin K; Vitamins

Topics: Animals; Naphthoquinones; Rabbits; Rats; Vitamin K; X-Rays

Topics: Diphosphates; Fibroblasts; Quinones; Sodium Chloride, Dietary; Tissue Culture Techniques; Vitamin K

Topics: Diphosphates; Sodium Chloride, Dietary; Vitamin K

Topics: Antifibrinolytic Agents; Naphthoquinones; Retinoids; Sodium; Vitamin K

Topics: Animals; Antifibrinolytic Agents; Chickens; Naphthoquinones; Vitamin K; Vitamin K 1; Vitamin K 3; Vitamin K Deficiency

Topics: Animals; Antifibrinolytic Agents; Naphthoquinones; Rats; Retinoids; Vitamin K; Water

Topics: Blood Coagulation; Blood Coagulation Tests; Factor V; Factor VII; Hemostatics; Humans; Liver; Liver Function Tests; Prothrombin; Prothrombin Time; Vitamin K; Vitamin K 1

Topics: Animals; Antifibrinolytic Agents; Naphthoquinones; Neoplasms, Experimental; Vitamin K; X-Rays

Topics: Animals; Antifibrinolytic Agents; Chickens; Hemostatics; Vitamin K; Vitamin K 1; Vitamin K 3; Vitamin K Deficiency

Topics: Animals; Characidae; Diphosphates; Drug Therapy; Neoplasms; Sodium; Vitamin K

Topics: Blood Coagulation; Dicumarol; Humans; Hypoprothrombinemias; Vitamin K; Vitamin K 1; Vitamin K 3

Topics: Dicumarol; Humans; Vitamin K; Vitamin K 1; Vitamin K 3

Topics: Dicumarol; Vitamin K; Vitamin K 1; Vitamin K 3