Morphological transformation by 8-hydroxy-2'-deoxyguanosine in Syrian hamster embryo (SHE) cells.
(41/863)
8-Hydroxy-2'-deoxyguanosine (OH8dG) is one of the most prevalent oxidative DNA modifications found in eukaryotic cells. Previous studies have suggested an association between OH8dG formation and carcinogenesis. However, it is unclear whether OH8dG formation results in the necessary genotoxic events for cancer development. In the present study, the formation of OH8dG and its ability to transform Syrian hamster embryo (SHE) cells was examined. Methylene blue, a photosensitizer that in the presence of light can generate singlet oxygen by a type II mechanism, was used to produce oxidative DNA damage (predominantly OH8dG) in SHE cells. Photoactivated methylene blue produced a dose-dependent increase in OH8dG as well as a dose-dependent increase in morphological transformation in SHE cells. SHE cells transfected with DNA that contained increasing concentrations of OH8dG displayed a dose-dependent increase in morphological transformation. Treatment with beta-carotene (a singlet oxygen quencher) inhibited both the formation of OH8dG and the induction of morphological transformation in photoactivated methylene blue-treated SHE cells. These results suggest that formation of OH8dG can induce morphological transformation and provide further support for a role of OH8dG formation in the carcinogenesis process. (+info)
Photodynamic treatment of pooled coumarin plasma for external quality assessment of the prothrombin time.
(42/863)
AIMS: To determine the conditions of photodynamic inactivation of vesicular stomatitis virus (VSV) added to pooled coumarin plasma and the effects of the photodynamic treatment on the prothrombin times and international normalised ratio (INR) in a Netherlands national external quality assessment scheme. METHODS: Pooled coumarin plasma samples were illuminated with visible light in the presence of 1 microM methylene blue. Inactivation conditions for VSV in pooled coumarin plasma were determined using an end point dilution assay. Plasma illuminated for 20 minutes was mixed with red blood cells and mailed to participants of the Netherlands external quality assessment (EQA) scheme. Prothrombin times and INRs were determined with various thromboplastin reagents. RESULTS: Photodynamic treatment using 1 microM methylene blue and 700 W/m2 caused 4.7 log inactivation of VSV in pooled coumarin plasma. Fibrinogen and coagulation factors II, V, VII, and X were decreased slightly by the treatment. These conditions caused prolongation of the prothrombin time in EQA surveys. The magnitude of the effect was different for various thromboplastin reagents. The increase of the INR was negligible when measured with the Thrombotest reagent. With other reagents, an approximately 5-16% increase of the INR was observed. Interlaboratory variation of the INR was not affected by photodynamic treatment. CONCLUSIONS: Photodynamic treatment of pooled coumarin plasma is very effective for the inactivation of some enveloped viruses such as VSV, but has only a limited effect on the prothrombin time and INR. Photodynamic treatment can be used to improve the viral safety of coumarin plasma for EQA of the prothrombin time and INR. (+info)
Stoichiometry of compounds bound to human erythrocytes in relation to morphology.
(43/863)
Most work on human erythrocyte interaction with drugs and other compounds has been reported on the basis of total concentrations. Total concentrations alone do not reveal numbers of molecules bound per cell, v. This paper emphasizes determination of v and of binding isotherms, in conjunction with changes in cell morphologies and in hypotonic shock behavior as v is varied. Four drugs and five other compounds were studied, with fresh erythrocytes. The principal findings are: (1) the intact erythrocyte engages in two kinds of binding mechanisms, statistical binding and cooperative binding, depending on the compound. In the case of a detergent, dodecylbenzene sulfonate, the binding is nearly quantitative. (2) The compounds often induce considerable protection against hypotonic hemolysis. However, the binding levels at which maximum protection occurs are rather close to the levels, vL, that occur upon complete conversion to the first distorted morphology. Therefore, the maximally protected erythrocyte may be a distorted erythrocyte. (3) The value n is the apparent total number of sites from Scatchard plotting for compounds which bind in a statistical manner. Levels vp and vw characterize maxima in cooperative binding behavior, also from Scatchard plotting of the data. Despite the wide diversity of over-all levels at which compounds exert their effects, the critical binding levels of and numbers of sites fall into a narrow range:n, vL, vP, and vw are all between 1 and 8 times 10-7 molecules or sites per cell. Most of our data, and that from some other laboratories, indicate that about 2 plus and minus 1 times 10-7 sites per erythrocyte are available for compound binding by the intact cell. Beyond that level, the cell in suspension almost always will be forced into the first obvious morphology change, as seen by phase contrast microscopy. (4) Once stoichiometries are established, the total binding capacity of erythrocytes for such compounds, in blood, can be estimated. An intruding organic molecule would encounter about 6 times as many plasma albumin sites as erythrocyte sites, if the plasma albumin sites were free. However, because albumin in vivo usually forms a complex with one to two fatty acids, the erythrocyte itself is rather likely to act as a transport particle for such compounds. (+info)
The relationship and significance of antibody titres as determined by various serological methods in glandular and ocular toxoplasmosis.
(44/863)
Three types of antibody curve have been demonstrated by testing sera during the course of acquired toxoplasmosis by six different techniques. These three types are due to cell-wall antibody, (demonstrated by four of the techniques), to antibody to soluble antigen, and to IgM antibody to the cell wall. These findings have been supported by absorption experiments. A scheme is presented for testing single sera by two or three different tests to indicate the stage and duration of the infection. (+info)
Diphenylene iodonium as an inhibitor for the hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct electron donor sites.
(45/863)
The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC- mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H2 as electron donor, Ph2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph2I and could be located on the distal [Fe4S4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe3S4] cluster and freely accessible from the periplasm. (+info)
An assay evaluation of the methylene blue method for the detection of anionic surfactants in urine.
(46/863)
Adding detergent to urine intended for drug testing is one of many ways to adulterate the specimen. This modified methylene blue procedure allows the detection and quantitation of anionic surfactants in urine. One-hundred urine specimens that exhibited normal foaming when shaken gave anionic surfactant values lower than 36 microg/mL with a mean of 8.73 microg/mL. Most of the suspected adulterated specimens and spiked samples with only 100 microL of detergent in 60 mL of urine had values greater than 750 microg/mL. Based on the analysis of negative samples, a urine specimen with an anionic surfactant level of 100 microg/mL or greater could be considered adulterated and most likely will have levels greater than 800 microg/mL. (+info)
Microsporidia and Candida spores: their discrimination by Calcofluor, trichrome-blue and methylene-blue combination staining.
(47/863)
Faeces of immunocompromised patients are often contaminated with the chitin-containing spores of microsporidia and Candida, which exclude the use of the chitin-specific fluorescent brightener Calcofluor white M2R for the identification of microsporidian spores. We developed a combination staining of Calcofluor white M2R with modified trichrome-blue staining and subsequent methylene-blue incubation which permits discrimination between these two types of spores. As a basis for diagnosis, a difference in the fluorescence pattern (365-440 nm) is combined with a difference in the light microscopic staining pattern. Under fluorescence conditions microsporidia spores have a spotted, brilliant white Calcofluor fluorescence and can easily be identified, while Candida spores show a reddish purple colour. Under the light microscope microsporidian spores show a light red colour with nonstained vacuole spots or strips in contrast to the yeast spores with their red-brown colour. This combination technique offers a highly specific means for the diagnosis of microsporidia spores in faeces. (+info)
Intrinsic and extrinsic light responses of Salmonella typhimurium and Escherichia coli.
(48/863)
Exposure to intense light in the region between 390 and 530 nm has been shown to have three effects on the motility of Salmonella typhimurium and Escherichia coli. Short pulses of light initiate continuous tumbling. Longer exposures to light induce smooth swimming, and prolonged exposures induced paralysis. The tumbling response is intimately connected with the chemical gradient-sensing apparatus of the bacterium and can be overcome by strong temporal gradients of attractant. Some mutants of S. typhimurium which are defective in the tumble-generating mechanism for chemotaxis are also unable to tumble in intense light. This intrinsic light effect can be mimicked by the addition of external dyes (the classical photodynamic effect), but it can be shown that the two phenomena are distinct. The extrinsic (photodynamic) effect can be inhibited by histidine or by anaerobic conditions, whereas the intrinsic effect is not. The observation that the extrinsic effect can also produce the three types of light responses listed above suggests a common pathway after an intial event on either an endogenous or an externally added photoreceptor. (+info)