Methylene blue plus light mediates 8-hydroxyguanine formation in DNA

Exposure to methylene blue (MB) plus light mediates formation of large levels of 8-hydroxyguanine in DNA. The amount of 8-hydroxy-2'-deoxyguanosine (8-OHdG) present in DNA increased as the amount of MB concentration increased throughout the 2 to 200 microM range studied and was dependent on light exposure. As the time of light exposure increased so did the 8-OHdG content to levels of about 750 8-OHdG/10(5) deoxyguanosine after 15 min of light exposure when MB was at 20 microM. Even though previous research has demonstrated that hydroxyl free radicals formed from a variety of sources mediate 8-OHdG formation in DNA, inclusion of mannitol, superoxide dismutase, catalase, and desferal in the MB plus light experiments demonstrated that these scavengers of oxygen free radical intermediates or precursors caused either no change or an increase in the 8-OHdG content of DNA exposed to MB plus light. These results appear to rule out the direct role of oxygen free radical intermediates in the primary events involved in the MB plus light mediated formation of 8-OHdG in DNA. Oxygen was essential to cause MB plus light mediated 8-OHdG formation in DNA. It was noted that when the reaction was carried out where the deuterium oxide content had been increased to 100%, the amount of 8-OHdG formed in DNA increased about threefold over that observed when comparable reactions were carried out in pure H2O. Use of the singlet oxygen scavenger 2,5-dimethylfuran has yielded variable results on the MB plus light mediated formation of 8-OHdG in DNA. The data taken collectively clearly indicate that MB plus light mediates 8-OHdG formation in DNA. The D2O data and the requirement for oxygen suggest that singlet oxygen may be an intermediate.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.     

Role of oxygen free radicals in carcinogenesis and brain ischemia

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin-trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage-induced loss of glutamine synthetase activity, may be a key event in stroke-induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8-hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin-traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.     

Derivatives of [3H]serotonin in organ cultures of hamster pineal gland

The purpose of this study was to determine the viability of the hamster pineal gland in organ culture and to test the effect of norepinephrine (NE) on [3H]serotonin derivatives. In this study, elevated levels of melatonin (7-fold, p less than .05), 5- hydroxytrytophol (5-fold, p less than .001), 5-methoxytryptophol (1.78-fold, p less than .05), and depressed levels of 5-hydroxyindoleacetic acid (3.8-fold, p less than .02) and methoxyindoleacetic acid (1.78-fold, p less than .05) were detected in the glands following the addition of NE to the medium. In a separate experiment, melatonin concentration in the media was also periodically measured by radioimmunoassay to determine the viability of the organ culture over a four-day period. The melatonin level on day 2 (2321 +/- 106 pg/gland) was significantly higher (p less than 0.01) than on day 3 (1542 +/- 86 pg/gland) or day 4 (805 +/- 39 pg/gland). The results of these experiments verify the viability of the hamster pineal organ culture and show that the gland responds to NE in vitro.     

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.     

The Response of Duck Erythrocytes to Hypertonic Media : Further evidence for a volume-controlling mechanism

The addition of a hypertonic bathing medium to duck erythrocytes results in an initial instantaneous phase of osmotic shrinkage and, when the [K]_o of the hypertonic solution is larger than "normal," in a second, more prolonged phase, the volume regulatory phase. During the latter, which also requires extracellular Na, the cells swell until they approach their initial isotonic volume. The increase in cell volume during the volume regulatory phase is accomplished by a gain in the cell content of K, Cl, and H₂O. There is also a smaller increase in the Na content of the cell. Potassium is accumulated against an electrochemical gradient and is therefore actively transported into the cell. This accumulation is associated with an increase, although dissimilar, in both K influx and efflux. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain, although this concentration of ouabain does change the cellular cation content. The response is independent of any effect of norepinephrine. The changes in cell size during the volume regulatory phase are discussed as the product of a volume controlling mechanism identical in principle to the one reported in the previous paper which controls cell volume in hypotonic media. Similarly, this mechanism can regulate cell size, when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

Investigation of respiratory and ion transport properties of aging bean stem slices

Ion and oxygen uptake were studied on aging bean stem slices. Oxygen uptake was high immediately after slicing, decreased to a minimum at 100 minutes, and then increased again. Ion uptake per unit of O₂ uptake data suggested that metabolic energy was utilized almost exclusively for sodium transport in fresh tissue but was diverted to potassium transport as the slices aged. Oxygen and ion uptake in fresh slices was less sensitive to 2,4-dinitrophenol as compared to the aged slices, indicating major metabolic and physiological changes occurred during aging. This was further substantiated by the tissue response to cyanide and antimycin A. Oxygen uptake was decreased by cyanide (22% by 1 mm) and antimycin A (14% by 1 microgram per milliliter) in fresh slices but not in aged slices. Potassium uptake that developed during aging was sensitive to cyanide and antimycin A. The results are pertinent to understanding the role of the stem in regulating ion transport in plants.