AUTOMATED MECHANICAL STIMULATION AND MEASUREMENT OF BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

A new integrated system for reproducible, automated mechanical stimulation and measurement of bioluminescence (BL) in multiple samples of marine dinoflagellate cell suspensions is described. The system was designed to allow the application of standardized experimental routines to parallel test vials for the purpose of toxicity testing. A sample tray delivered test vials to the position of mechanical stimulation and BL measurement. Mechanical stimulation of BL was applied as sharp rotation-onset of the test vial about its vertical axis. Thus, any direct chemical or physical perturbation of the cell suspension was avoided. A silicon photovoltaic cell measured the emitted light. Stimulation, measurement and recording of BL were integrated and controlled by specially developed software, which runs on a personal computer in the graphic environment of MS-Windows. Precise scheduling, flexible programming and identical repetition of experimental routines are possible in practice. For Gonyaulax polyedra, details of BL, as stimulated and measured with the new system, are presented and discussed. We conclude that the system exhibits specific features that offer wide potential of application in several fields of research on dinoflagellate BL, particularly for toxicity testing.     

ACTION SPECTRUM FOR THE PHOTOINHIBITION OF BIOLUMINESCENCE IN THE MARINE DINOFLAGELLATE DISSODINIUM LUNULA

Abstract— The fractional photoinhibition of the mechanically stimulable bioluminescence in the vacuolar dinoflagellate Dissodinium lunula is proportional to the logarithm of the exposure. The action spectrum for this photoinhibition has been determined by measuring threshold exposures in absolute units of photons cm⁻². The threshold exposure at the wavelength of maximum sensitivity, 450 nm, was 2 ± 10⁻² photons cm⁻². The action spectrum is consistent with absorption by a blue light receptor pigment shielded by a nonphotoactive pigment which absorbs in the region of the bioluminescence emission spectrum. It is suggested that there may be some selective advantage for this absorbing pigment in the vacuolar dinoflagellates in order to prevent the organisms from being photoinhibited by their own bioluminescence.     

THE EVOLUTION OF BIOLUMINESCENCE IN BACTERIA

Individuals of aerobic, saprophytic bacterial species, encountering a changing environment in which they were regularly exposed to periods of severe hypoxia, would have gained a major selective advantage in fatty acid metabolism if a mutation in a flavoprotein oxygenase permitted them to oxidize accumulated C₈-C₁₈ aliphatic aldehydes to fatty acids at oxygen concentrations below which the cytochrome oxidase mediated electron transport pathway became inhibited. Such mutants, able to continue to metabolize exogenous lipid fatty acids under hypoxia, might have outproduced their wild type ancestors. Colonies of those mutants which utilized reduced flavin mononucleotide (FMNH₂) as the flavin cofactor would have been luminous, owing to the fortuitous coincidence that the aldehyde oxygenation resulted in an enzyme-flavin excited electronic state which emitted blue light with a high fluorescence yield. If this accidental “proto-bioluminescence” were initially of sufficient brightness to have elicited phototactic responses in nearby motile organisms, increasing thereby detrital food sources or the potential for dispersal and colonization for the bacteria, a new and completely different selective advantage, that of bioluminescent signalling, would have arisen from the original metabolic function. This is the biochemical analog of Darwin's principle of functional change in structural continuity. It is proposed that bacterial luciferase, the FMNH₂-oxygenase in luminous bacteria, was such a mutation. The present ubiquitous distributions of luminous bacterial species in marine waters, on the surfaces of marine animals and as symbionts in the specialized light organs and digestive tracts of many fish species have resulted from subsequent environmental selection and optimization for this original proto-bioluminescent reaction. By extension it is suggested whereas “protobioluminescence” arose in many species independently whenever metabolic oxygenation of a substrate resulted in an adventitiously efficient chemiluminescence, the function of bioluminescence arose only upon favorable interaction between the “proto-bioluminescence” and its ecosystem.     

PHOTOINHIBITION OF PHOTOSYNTHESIS IN NATURAL WATERS*

Abstract— A quantitative analysis of the wavelength-dependent influence of solar irradiance on natural phytoplankton photosynthesis has been made. The effect on productivity due to several different UV radiation regimes has been measured. In the course of this analysis, it has been shown that the biological weighting function for photoinhibition of chloroplasts (Jones and Kok, 1966) allows the calculation of a biologically effective dose which is consistent with the measured photoinhibition in natural phytoplankton populations. The ecological implications of a change in available UV radiation, possibly due to anthropogenic altering of the ozone layer, are explored and it is found that the present static bottle ^l4C technique of measuring in situ phytoplankton productivity does not lend itself to assessing accurately the potential ecological consequences of possible increased MUV (middle ultraviolet radiation in the 280–340 nm region) on phytoplankton populations. A small change in MUV has a relatively minor effect on photoinhibition dose rates whereas it has a large potential effect on DNA dose rates.     

PHOTOINHIBITION OF CHLOROPLAST REACTIONS

Abstract— An attemlpt was made to localize the site of photoinhibition of photosynthesis by measuring the decay of various chloroplast reactions after exposure to very strong light. A11 substrate reductions coupled to oxygen evolution as well as photophosphorylation mediated by PMS, proved equally sensitive to photoinhibition. Reactions involving only the long wave photosystem, such as TPN reduction with ascorbate as electron donor and photooxidation of cytochromec by detergent-treated chloroplasts were sensitive to a lower degree.
Photoinhibition irreversibly annihilated the 'variable' fraction of fluorescence emission —it decreased the steady state yield 2-3 fold and abolished the slow rise of the emission at the onset of illurnination.
It is concluded that the primary site of light inactivation is in, or close to, the trapping centers of the oxygen evolving step of photosynthesis. Pre-illumination leaves these traps in a state capable of draining light from sensitizing pigments but unable to perform useful photochemistry.     

INVOLVEMENT OF THYLAKOID MEMBRANE-DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

Photoinhibition of the light-regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light-induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (¹O_2,¹Δ_g) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light-induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe-S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by ¹O₂, which is photoproduced primarily by the Fe-S centers of thylakoids and diffuses into the stroma.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

INDUCTION OF BIOLUMINESCENCE IN THE MARINE FISH, Porichthys, BY Vargula (CRUSTACEAN) LUCIFERIN. EVIDENCE FOR de novo SYNTHESIS OR RECYCLING OF LUCIFERIN

Abstract— The marine fish, Porichthys notatus , emits light by a classical luciferin-luciferase reaction whose components are similar, if not identical, to those found in the luminescent crustacean, Vargula. Porichthys is divided geographically into a southern luminescent and a northern nonluminescent population. Specimens of nonluminescent Porichthys can be induced to become luminescent by injection or ingestion of Vargula luciferin. After feeding a known quantity of Vargula luciferin, light emitted by Porichthys was monitored for a 2-yr period. Summation of light produced during each bioluminescence episode demonstrated that the total quanta emitted over 2 yr exceeded the theoretical yield from the administered luciferin. These results indicate that the administered luciferin either recycles or induces de novo synthesis of additional luciferin in Porichthys .     

BIOCHEMISTRY OF CENTIPEDE BIOLUMINESCENCE*

Abstract— The centipede (Orphaneous brevilabiatus) secretes a bioluminescent slime. The corrected emission spectrum of this luminescence was found to have maxima at about 510 and 480 nm. The reaction was found to require both a luciferin and luciferase and showed an unusually low pH optimum (4.6). Oxygen was required for the reaction, but oxygen could interact with one of the components allowing for anaerobic light emission.     

THE ORIGIN OF BIOLUMINESCENCE*

Abstract— Primitive luciferases evolved in order to utilize oxygen directly as an electron acceptor at the low oxygen concentrations of the primitive atmosphere. This provided a major selective advantage in the ability to metabolize aromatic molecules and n-alkanes. These strongly exergonic reactions produce product molecules in electronically excited states, from which light emission is possible. The low-level luminescence observed from microsomal extracts, from the action of leukocytes on phagocytized bacteria and from rapidly growing tissues is ascribed to these same exergonic hydroxylase reactions, or to the release during these reactions of superoxide radicals which can initiate chemiluminescent reactions. Bioluminescence is a later secondary adaptation of this oxygenase reaction to signaling for sex, food or escape. The similarity of the reaction pathways is shown in the three bioluminescent systems from which the substrates and products have been identified.     

火焰原子吸收光谱法快速测定海水沉积物中总铬

利用火焰原子吸收光谱法直接测定海水沉积物中总铬。方法的特征浓度为0.045 mg.L-1(1%吸收),检出限为0.030 mg.L-1,相对标准偏差为2.2%~5.8%,加标回收率为86.4%~98.6%。     

ANALOGS AND DERIVATIVES OF FIREFLY OXYLUCIFERIN, THE LIGHT EMITTER IN FIREFLY BIOLUMINESCENCE

Abstract— The 5-methyl analog of firefly oxyluciferin, two isomeric O-methyl ether derivatives of it and an O, O Ó-dimethyl ether derivative were synthesized and their UV absorption and fluorescence emission spectra were determined. Comparisons of the emission data with the emission wavelength in bioluminescence indicate that the mono-anions of firefly oxyluciferin are candidates for the light-emitters in bioluminescence. Further, we have found that the chemiluminescence of active esters of firefly luciferin produces (from the keto form of oxyluciferin) only red light emission under a variety of conditions; a yellow-green light emission (from the enolic forms of the oxyluciferin product) could not be elicited.     

两种原子荧光法测定不同类型海洋沉积物中痕量汞

采用流动注射 冷蒸气发生 无色散原子荧光光谱法 ,以硼氢化钾为还原剂 ,不经富集直接测定了近海海洋沉积物标准物质和不同类型海洋沉积物中痕量汞 ,分析准确度和精密度良好 ,所测样品中汞含量范围为 0 .0 0 6～ 0 .172 μg·g- 1;并用冷原子荧光光度法以氯化亚锡为还原剂测定相同的样品 ,两组数据符合良好。两种原子荧光法的工作曲线 (汞含量为 0 .0 0 0～ 1.0 0 0 μg·L- 1)相关系数分别为 0 .9997和 0 .9999,在海洋沉积物中汞的检出限分别为 0 .4和 0 .2ng·g- 1。     

ENERGY TRANSFER VIA PROTEIN-PROTEIN INTERACTION IN RENILLA BIOLUMINESCENCE

Abstract—Radiationless energy transfer is known to play biologically important roles in both photosynthesis and bioluminescence. In photosynthesis, accessory pigments serve as "antennae", transferring excitation energy into the "reaction centers". In the bioluminescent coelenterates, energy is transferred from the site of reaction via an accessory protein known as the green-fluorescent protein (GFP). Coelenterate bioluminescence systems such as that of the sea pansy, Renilla , are well characterized biochemically, and their energy transfer process can be duplicated in vitro using isolated and purified components. We have measured efficient in vitro energy transfer from the electronic excited state of the enzyme-bound oxyluciferin to the green-fluorescent protein at protein concentrations of 0.1 μ M . We have also demonstrated a 1:l complex between these proteins, under conditions of energy transfer, by the chromato-graphic technique of Hummel and Dreyer. These observations indicate that bioluminescent energy transfer is mediated via protein-protein interaction. Furthermore, with inter-species cross-reaction studies and protein modification techniques we have shown that the interaction between luciferase and GFP is highly specific. These features make the Renilla system an attractive alternative to the photosynthetic systems as a tool for studying radiationless energy transfer.     

APPLICATION OF PHOTOSENSITIVE DEVICES TO BIOLUMINESCENCE STUDIES

Abstract—A brief review is given of some results obtained by the application of image intensification to studies of bioluminescence. The system consists of an image intensifier placed at the output of a suitable microscope., so that the image from the microscope falls on the intensifier cathode. The photon gain of the intensifier can be varied from a few thousand to one million. The output of the intensifier is recorded either on film or, in most applications to date, by means of a TV vidicon. The TV system permits display on a monitor in real time and simultaneous recording on magnetic tape for subsequent playback and analysis. It also provides time resolution for dynamic studies. Results are summarized for in vivo observations on Noctiluca miliaris, Obelia, Renilla , and Mnemiopsis leidyi . Utilization of the luminescence of aequorin in the presence of Ca²⁺ has been directed to observations on amoebae and the egg of the Medaka fish.
Studies at the molecular level have been made by means of the spectral distribution of the output light. In these, the output of a fast input lens grating spectrometer is focused on the image intensifier cathode. Thus the entire visible spectrum of an in vivo bioluminescent flash can be intensified and recorded on film by photographing the output. The film is then analyzed by means of a digitized densitometer, and a computer program corrects the observed spectrum for system non-linearities and non-uniformities. In this way, the in vivo spectra of 15 bioluminescent species have been recorded.