Thérèse Wilson

Reactive oxygen species as cellular messengers

Reactive oxygen species (ROSs) have recently been found to be important signaling molecules in several cellular responses. Individual species have characteristic reactive properties, yet are easily interconverted, making it difficult to identify the ROSs involved in each response.

CAROTENOID CHROMOPHORE LENGTH AND PROTECTION AGAINST PHOTOSENSITIZATION

Abstract— Carotenoid pigments were extracted and purified from wild‐type and mutants 7 and 93a of Sarcina lutea, and tested for their ability to quench 1O2. The wild‐type pigment (P‐438, 9 conjugated double bonds) is as active in quenching 1O2 as is β‐carotene. On the other hand, the pigment P‐422 (8 conjugated double bonds) from mutant 7 is 2 or 3 times less efficient, whereas phytofluene and phytoene from S. lutea are 100 and 1000 times less efficient, respectively, than is β‐carotene at quenching 1O2. It was also found that the broad EPR signal, induced by light in benzene solutions of chlorophyll a and hydroquinone, and related to chlorophyll oxidation, is efficiently quenched by P‐438 and to a much smaller extent also by Sarcina phytoene.

COMMENTS ON THE MECHANISMS OF CHEMI- AND BIOLUMINESCENCE

Although there is an astonishing diversity of bioluminescent organisms, from bacteria and fungi to mollusks, crustaceans, insects and fishes, there is a degree of unity at the chemical level: bioluminescences are enzyme-catalyzed reactions of molecular oxygen with appropriate substrates, and in all probability all involve the formation and breakdown of a peroxide or hydroperoxide. Going one step further, one might classify the bioluminescence systems into two groups, those that are considered to proceed via the intermediacy of a four-member ring peroxide and those that involve linear peroxides. I will mainly review here what has been learned from chemiluminescence regarding the first group, focusing on still unanswered questions that bear also on the mechanism of bacterial bioluminescence, the foremost example of a system involving a linear peroxide. The discussion will attract attention to a mechanistic dilemma posed by the widely quoted CIEEL* (chemically initiated electron exchange luminescence) proposal.

Two Bioluminescent Diptera: The North American Orfelia fultoni and the Australian Arachnocampa flava. Similar Niche, Different Bioluminescence Systems¶

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (λmax= 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin–luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of ∼140 kDa and an additional high molecular weight fraction (possibly a luciferin‐binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin–luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.

A blue fluorescent protein from a yellow-emitting luminous bacterium

Abstract— Vibrio fischeri strain Y1 emits yellow light in vivo due to the participation of a yellow fluorescent protein (YFP) in the luciferase reaction. In this study it was found that the organism also produces a protein (referred to as Y1‐BFP) emitting strong blue fluorescence. Its molecular weight, about 25 kDa, is the same as or very close to that of YFP. The fluorescence excitation and emission maxima of the purified Y1‐BFP are at 416 and 461 nm, respectively, and the fluorescence lifetime is 12.5 ns at 2°C. The molar extinction coefficient of Y1‐BFP at 416 nm was estimated to be approx. 9500. With the homologous luciferase, Y1‐BFP decreases the intensity and rate of decay in the in vitro reaction but has no effect on its emission spectrum (in contrast to YFP, which has a striking effect on the spectrum). With luciferase isolated from Vibrio harveyi, however, Y1‐BFP causes a small blue‐shift (˜10 nm) in the emission of the enzyme catalyzed reaction, whereas YFP has no effect on the emission spectrum.

Two water soluble fluorescence probes for chemiexcitation studies sodium 9 10 dibromo and 9 10 diphenylanthracene 2 sulfonate synthesis properties and application to triplet acetone and tetramethyldioxetane

The syntheses of sodium 9,10‐dibromo‐ and 9,10‐diphenylanthracene‐2‐sulfonate (DBAS and DPAS, respectively) are described and their photophysical properties determined. These two probes were used in aqueous solution studies of the kinetic parameters of tetramethyldioxetane thermolysis, which were found to be the same as in organic solvents. The yields of triplet and singlet acetone generated by the decomposition of this dioxetane in water are also comparable to the literature values in organic medium. The lifetime of triplet acetone in water was determined to be 13 ± 2 u.s by a method based on the measurement of the fluorescence decay of DBAS excited via energy transfer from triplet acetone, by the time‐correlated single‐photon counting technique. Sorbate ion quenches triplet acetone from tetramethyldioxetane with a rate constant smaller but close to the diffusion‐controlled limit.

CHEMILUMINESCENCE FROM THE ENDOPEROXIDE OF 1,4-DIMETHOXY-9,10-DIPHENYLANTHRACENE

INTEREST in the chemiluminescent decomposition of polyacene endoperoxides [ 11 derives from the early work of Dufraisse and his co-workers[2], who identified the products as molecular oxygen and the original polyacene. This apparent overall reversibility of the photooxygenation process, a typical singlet oxygen reaction, can reach 98% for the endoperoxide (11) of 1,4-dimethoxy-9,10-dipheny1anthracene (I) which is the most chemiluminescent [3] of the anthracene derivatives.

SPECTRAL OBSERVATION OF SINGLET MOLECULAR OXYGEN FROM AROMATIC ENDOPEROXIDES IN SOLUTION

Abstract— The characteristic near‐infrared emission band of O2 (1Δg) at 1.28 μm has been recorded from carbon tetrachloride solutions of the 1, 4‐endoperoxides of 1,4‐dimethyInaphthalene and 1, 4‐dimethoxy‐9.10‐diphenylanthracene undergoing thermal decomposition.

PASSIVE AND ACTIVE ROLE OF FLUORESCERS IN THE CHEMILUMINESCENCE OF TETRAMETHOXYDIOXETANE

Abstract— Tests for the possible catalytic role of fluorescers in the chemiluminescent decomposition of dioxetanes are discussed and applied to the case of tetramethoxydioxetane. This dioxetane, which can undergo direct thermal decomposition yielding excited methyl carbonate with subsequent excitation of fluorescers (for example 9, 10‐dibromoanthracene. DBA), is shown to be sensitive also to catalysis by fluorescers such as rubrene. 9, 10‐diphenyl‐anthracene (DPA) and 9, IOdicyanoanthracene (DCNA). The possibility of a charge‐transfer mechanism of chemiluminescent catalysis is suggested.

A transcriptional and proteomic survey of Arachnocampa luminosa (Diptera: Keroplatidae) lanterns gives insights into the origin of bioluminescence from the Malpighian tubules in Diptera

Fungus-gnats of the genus Arachnocampa are unique among bioluminescent insects for displaying blue-green bioluminescence, and are responsible for one of the most beautiful bioluminescence spectacles on the roofs of the Waitomo Caves. Despite morphological studies showing that Arachnocampa larval lanterns involve specialization of the Malpighian tubules, the biochemical origin of their bioluminescence remains enigmatic. Using a cDNA library previously constructed from lanterns of the New Zealand glowworm A. luminosa, we carried out the first transcriptional analysis of ~ 500 expressed sequence tags (ESTs) to identify putative candidate proteins for light production, and to better understand the molecular physiology of the lanterns and their relationship with Malpighian tubule physiology. The analysis showed an abundance of hexamerin-like proteins, as well as luciferase-like enzymes, indicating a possible critical role for these proteins in bioluminescence. These findings were corroborated by proteomic analysis of lantern extracts, which showed the presence of hexamerins and luciferase-like enzymes. Other gene products typical of Malpighian tubules, such as detoxifying enzymes, were also found. The results support the existence of an evolutionary link between Malpighian tubule detoxification and the origin of bioluminescence in these Diptera.