New developments in millimeter wave spectroscopy

In the past few years millimeter and submillimeter wave spectroscopy has been the backbone for modern molecular line astronomy. The detection of unstable and high-temperature molecular species in the interstellar environment has stimulated the development of methods of detecting molecular ions, radicals and unstable molecules in the laboratory by means of millimeter wave technology. Some of the recent experimental advances as well as the developments which have occurred in the analysis of high resolution rotational and vibrational spectra with regard to understanding molecular dynamics will be discussed.     

Chemical Analysis of the Luminous Slime Secreted by the Marine Worm Chaetopterus (Annelida,Polychaeta)

The marine annelid Chaetopterus variopedatus produces bioluminescence by an unknown and potentially novel mechanism. We have advanced the study of this fascinating phenomenon, which has not been investigated for nearly 60 years after initial studies were first reported for this species. Here, we show that the luminous slime produced by the worm exhibits blue fluorescence that matches the bioluminescence emission. This result suggests that the oxyluciferin emitter is present. However, while the blue fluorescence decays over time green fluorescence is increasingly revealed that is likely associated with products of the luminescence reaction. LC/MS and fluorescence analysis of harvested luminescent material revealed riboflavin as the major green fluorescent component. Riboflavin is usually associated with the mechanism of light production in bacteria, yet luminous bacteria were not found in the worm mucus, and accordingly were not reported to be directly responsible for the light emission, which is under nervous control in the worm. We therefore propose a hypothesis in which riboflavin or a structurally related derivative serves as the emitter in the worm's light producing reaction.     

Synthesis and luminescence properties of biphenyl-type firefly luciferin analogs with a new,near-infrared light-emitting bioluminophore

New firefly luciferin analogs of the 4,4′-substituted biphenyl-type were synthesized. One analog with a 4′-dimethylamino group possessed bioluminescence activity, emitting near-infrared biological window light at 675 nm suitable for deep-site bioimaging of living animals. The chemiluminescence light-emission maximum of the corresponding methyl ester of the bioluminescence active analog was 500 nm, implying that biphenyl and thiazolinone rings in the light emitter might be placed in a coplanar conformation at the polar luciferase active site.     

DIMENSIONAL PROBING OF THE ATP BINDING SITE ON FIREFLY LUCIFERASE

Abstract— lin -Benzoadenosine 5'-triphosphate has been shown to be an acceptable substrate for light production in the firefly luciferase-luciferin system. This nucleotide analogue displays strong enzyme binding and a reduced rate of enzyme catalysis compared with ATP. Variation in the color of the bioluminescence emission with /in-benzo-ATP compared with ATP suggests that a lateral extension in the purine base induces a change in the conformation of the luciferase and in the environment of the excited light emitter.     

Bioactivity screening by chromatography-bioluminescence coupling

Summary Because luminescent microorganisms change their light emission in the presence of bioactive substances, they provide an effective way of monitoring toxicity. Here we report the direct coupling of bioluminescence to chromatography which is capable of detecting single toxic components in complex mixtures. Two approaches were investigated, utilizing thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The TLC-bioluminescence imaging technique was developed into a versatile and rugged method and proved to be superior to the HPLC-based design. Employing genetically engineered luminescent bacteria gives customized selectivity of bioactivity detection. The combination of analytical separation technology with the biospecific sensing ability of living cells provides a novel screening tool for targeting bioactive components in complex samples.     

Marine Bioluminescence: Measurement by a Classical Light Sensor and Related Foraging Behavior of a Deep Diving Predator

Bioluminescence is produced by a broad range of organisms for defense, predation or communication purposes. Southern elephant seal (SES) vision is adapted to low‐intensity light with a peak sensitivity, matching the wavelength emitted by myctophid species, one of the main preys of female SES. A total of 11 satellite‐tracked female SESs were equipped with a time‐depth‐light 3D accelerometer (TDR10‐X) to assess whether bioluminescence could be used by SESs to locate their prey. Firstly, we demonstrated experimentally that the TDR10‐X light sensor was sensitive enough to detect natural bioluminescence; however, we highlighted a low‐distance detection of the sensor. Then, we linked the number of prey capture attempts (PCAs), assessed from accelerometer data, with the number of detected bioluminescence events. PCA was positively related to bioluminescence, which provides strong support that bioluminescence is involved in predator–prey interactions for these species. However, the limitations of the sensor did not allow us to discern whether bioluminescence (i) provided remote indication of the biological richness of the area to SES, (ii) was emitted as a mechanic reaction or (iii) was emitted as a defense mechanism in response to SES behavior.     

Yellow-green and red firefly bioluminescence from 5,5-dimethyloxyluciferin

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) similar 530 nm) to red (lambda(max) similar 635 nm). The original mechanism of bioluminescence color determination advanced by White and co-workers was based on the concept that the keto and enol tautomers of the emitter oxyluciferin produce red and green light, respectively. Alternatively, McCapra proposed that color variation is associated with conformations of the keto form of excited-state oxyluciferin. We have prepared the adenylate of D-5,5-dimethylluciferin and shown that it is transformed into the putative emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the green-emitting click beetle. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, may be taken as the first experimental support for McCapra's mechanism of firefly bioluminescence color or any other proposal that requires only a single keto form of oxyluciferin.     

Identification of mutant firefly luciferases that efficiently utilize aminoluciferins

Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin‐binding domain and streptavidin, with proteins A and G, antibodies, with DNA‐ and RNA‐binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase‐based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET‐based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein–protein interactions, assaying of metabolites involved in cell communication and cell signaling.     

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.     

EVIDENCE FOR HIGH ENERGY STORAGE INTERMEDIATES IN BIOLUMINESCENCE

Abstract— The bioluminescent enzyme from Photobacterium fischeri is normally activated in vifro by reaction with FMNH₂ and O₂. in the presence of a long-chain aldehyde. Emission from enzyme intermediates in this reaction continues for several seconds, and if the mixture is frozen just after initiation of the reaction, this presumptive emission may be delayed until the system is warmed again. Light is then emitted in a fashion analogous to thermo-luminescent emission, with a maximum intensity at — 10°C. The experiments described here show that the total amount of light which is emitted under these conditions no longer depends so much upon aldehyde, a relatively high quantum yield being obtained both with and without aldehyde.
It is further shown that bioluminescence may be activated by light, populating it is believed, the same state which is responsible for the emission in the case of the FMNH₂-induced emission. The light-induced reaction does not depend on flavin in the enzyme preparations, nor does the activation spectrum resemble that of a flavoprotein. Activation may be carried out in the solid state at temperatures down to at least — 100°C, and so does not involve the diffusion of large molecules. It is proposed that energy storage takes place by charge separation, and that the excited state from which emission takes place is associated with charge recombination.     

发光细菌传感器在纳米氧化物紫外屏蔽性能评估分析中的应用研究

本文发现发光细菌在紫外光照射下能够被杀死，其荧光强度相应地发生降低，而在纳米氧化物的保护下，细菌荧光强度的降低得到了抑制，因此发光细菌可以被用来分析和评价纳米氧化物的紫外屏蔽性能。青海弧菌Q67是发光细菌的一种，本文研究了不同浓度及不同种类的纳米氧化物对本发光细菌在分别受到UVA、UVB、UVC紫外光照射下的发光强度的影响，根据细菌发光强度降低的相对值建立了一种分析和评价纳米氧化物的紫外屏蔽性能的方法。该方法可以对纳米氧化物在UVA、UVB、UVC区的紫外屏蔽性能进行评价，同时也为化妆品等行业提供了一种对防晒剂紫外屏蔽性能评估的有效方法。     

NAPHTHYL- AND QUINOLYLLUCIFERIN: GREEN AND RED LIGHT EMITTING FIREFLY LUCIFERIN ANALOGUES

In the course of investigations on the possible involvement of the CIEEL (chemically initiated electron-exchange luminescence) mechanism in firefly bioluminescence, we have synthesized two novel firefly luciferin substrate analogues. D-Naphthylluciferin and D-quinolylluciferin were prepared by condensing D-cysteine with 2-cyano-6-hydroxynaphthalene and 2-cyano-6-hydroxyquinoline, respectively. These analogues are the first examples of bioluminescent substrates for firefly luciferase that do not contain a benzothiazole moiety. Firefly luciferase-catalyzed bioluminescence emission spectra revealed that compared to the normal yellow-green light of luciferin (lambda max = 559 nm), the emission from naphthylluciferin is significantly blue-shifted (lambda max = 524 nm); whereas quinolylluciferin emits orange-red light (lambda max = 608 nm). The fluorescence emission spectra, reaction pH optima, relative light yields, light emission kinetics and KM values of the analogues also were measured and compared to those of luciferin. Neither of the analogues produced the characteristic flash kinetics observed for the natural substrate. Instead, slower rise times to peak emission intensity were recorded. It appears that the formation of an intermediate from the analogue adenylates prior to the addition of oxygen is responsible for the slow rise times. The synthetic substrate analogues described here should be useful for future mechanistic studies.     

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.     

Coherent radiation from high-current electron beams of linear accelerators and its applications

The characteristics of the far-infrared light source using the coherent radiation emitted from a high-energy short electron bunch have been investigated. The coherent radiation has a continuous spectrum in a submillimeter to millimeter wavelength range and the brightness is relatively high. The spectrum of the radiation is determined by the longitudinal form factor of the electron bunch. The operational conditions of a high-current linear accelerator have been optimized using an electron bunch shape monitor. The coherent transition radiation light source has been applied to absorption spectroscopy for liquid water and to an imaging experiment for a leaf of rose.     

On the requirement and mode of action of long-chain aldehydes during bacterial bioluminescence

Abstract— We have reinvestigated the role of aldehyde in bacterial bioluminescence relative to its absolute requirement for light emission. We have found that aldehyde is an absolute requirement for light emission at 25°C as well as in the frozen state (—3° to — 9°C). As found by earlier workers, certain luciferase preparations isolated from Ph. Jischeri do not appear to require aldehyde for bioluminescence from the frozen state. We can now attribute this behavior to contaminating levels of aldehyde in those preparations. The results suggest that reactant puddling exist in the frozen state in which micro regions of liquid form within the ice crystals resulting in enormous increases in reactant concentration.     

Imaging Macrophage Phagocytosis Using AIE Luminogen‐Labeled E. coli

Phagocytosis of bacteria is an important biological process. Gaining insight into this process may greatly benefit related pathological studies and further contribute to development of therapies for infectious diseases. Tools for studying these internalization processes, however, are limited. Herein, we demonstrate the feasibility of employing an environmentally sensitive aggregation‐induced emission (AIE) probe for bacteria labeling and imaging. By tracking the fluorescence variation of the stained bacteria, the pH changes of its microenvironment can be monitored. In this way, the phagocytic entry of these bacteria into the macrophage cells and the intravacuolar acidification can be visualized in real‐time.     

Theoretical Study of Dinoflagellate Bioluminescence

Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin–luciferase one. However, the excited‐state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin–luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.