VARIATION IN THE ULTRASTRUCTURE OF THE BIFLAGELLATE MOTILE CELLS OF SIX UNICELLULAR GENERA OF THE CHLAMYDOMONADALES AND CHLOROCOCCALES (CHLOROPHYCEAE), WITH EMPHASIS ON THE FLAGELLAR APPARATUS

The ultrastructural features of the biflagellate motile cells of six different species of the Chlorophyceae, namely Dunaliella lateralis (Polyblepharidaceae, Chlamydomonadales), Chlorococcum hypnosporum, Spongiochloris spongiosa, Protosiphon botryoides (Chlorococcaceae, Chlorococcales), Tetracystis aeria and Pseudotetracystis terrestris (Tetracystidaceae, Chlorococcales), were examined with an emphasis on the flagellar apparatus (FA). They have different vegetative characteristics, such as, being motile or nonmotile, variations in chloroplast morphology, possession of one or more nuclei, and reproductive features such as formation of tetrahedral tetrads, and naked or walled zoospores. Ultrastructural differences amongst reproductive cells of the six species include variations in cell surface structure, basal body to basal body angle, beamlike extensions of the distal fiber, extensive connections of the proximal sheath between basal bodies, two-membered rootlets, striated microtubule-associated components, two-membered rootlet-nucleus and/or mitochondria connections, X-membered rootlets, connections of rootlets and basal bodies, rhizoplasts and accessory basal bodies. All six species possess pyrenoids penetrated by thylakoid membranes, and the FA typical of the Chlorophyceae (sensu Mattox and Stewart, 1984). These six species should be divided into two groups. The first includes D. lateralis, C. hypnosporum, and T. aeria, in which accessory basal bodies are present, the basal body to basal body angle is relatively fixed, and a cell wall or surface coat is present. The second group includes Ps. terrestris, S. spongiosa, and Pr. botryoides, in which accessory basal bodies are absent, the basal body to basal body angle is variable and the zoospores are naked.     

Increased 8-hydroxyguanine content of chloroplast DNA from ozone-treated plants

The mechanism of ozone-mediated plant injury is not known but has been postulated to involve oxygen free radicals. Hydroxyl free radicals react with DNA causing formation of many products, one of which is 8-hydroxyguanine. By using high performance liquid chromatography with electrochemical detection, the 8-hydroxy-2′-deoxyguanosine (8-OHdG) content of a DNA enzymatic digest can be sensitively quantitated. Beans (Phaseolus vulgaris L.) and peas (Pisum sativum L.) were treated with an ozone regime that caused acute injury. Chloroplast DNA was obtained from plants harvested either immediately after ozone treatment or 24 hours later. Ozone-exposed plants in general had nearly two-fold higher levels of 8-OHdG as compared to control plants. In vitro treatment of DNA in buffer solution with ozone did not cause formation of 8-OHdG in DNA, even though ozone did react directly with the macromolecule per se. Exposure of isolated, illuminated chloroplasts to ozone caused nearly a seven-fold increase in the amount of 8-OHdG in the chloroplast DNA as compared to none-ozone-exposed chloroplasts. These results suggest that ozone exposure to plants causes formation of enhanced levels of oxygen free radicals, thus mediating formation of 8-OHdG in chloroplast DNA. The reaction of ozone with DNA per se did not cause formation of 8-OHdG. Therefore, it is the interaction of ozone with plant cells and isolated chloroplasts which mediates oxygen free radical formation.     

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.