Synthesis of dinucleoside tetraphosphates in transfected cells by a firefly luciferase reporter gene

The firefly luciferase gene is widely used as a reporter gene and its expression is generally considered to be non-toxic. In addition to its light-producing reaction, luciferase can synthesise dinucleoside polyphosphates, intracellular signalling molecules, in vitro. Here we show that COS-7 cells transfected with a luciferase expression vector accumulate up to 0.5 mM adenine-containing dinucleoside tetraphosphates (Ap₄N) during the 24 h following luciferin addition. The optimal external concentration of luciferin was 0.4–0.6 mM. In agreement with its poor ability to synthesise adenine-containing dinucleoside triphosphates in vitro, the level of these compounds did not increase after transfection. Consequently, the results of experiments involving luciferase-mediated light production by live cells should now be viewed in the light of the possible effects of an increased intracellular Ap₄N concentration on the properties of the system under investigation. This observation also points to a useful non-invasive procedure for the specific enhancement of intracellular Ap₄N for studies directed at understanding the functions of these compounds.Received 12 November 2003; received after revision 10 December 2003; accepted 12 December 2003     

Inhibition of firefly bioluminescence by scavengers of singlet oxygen,superoxide radicals and hydroxyl radicals

Summary The participation of highly energetic oxygen species in the ATP-induced bioluminescence of a firefly-extract has been investigated. The inhibition of light emission by a variety of specific scavengers suggests that singlet oxygen, superoxide radicals and hydroxyl radicals are important intermediates in the firefly bioluminescence reaction.Acknowledgments. I thank Prof. R. Bachofen, Institut für Allgemeine Botanik, Abteilung Mikrobiologie, Universität Zürich, Switzerland, in whose laboratory most of these studies have been performed, for his cooperativity. Financial support by the Deutsche Studienstiftung is gratefully acknowledged.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Effects of larval firefly extracts on molluscan cardiac activity

Summary Larval firefly midgut extracts and larval firefly hemolymph extracts were found to produce a potent inhibitory effect when applied directly to the heart of a terrestrial mollusc. It is suggested that such substances could comprise an important part of the paralyzing toxin which is reportedly injected by larval fireflies into their prey.This work was supported by The Graduate School, University of Wisconsin-Milwaukee.     

Further measurements on the bioluminescence of the seedlings

Riassunto Alcuni di noi recentemente hanno messo in evidenza un 'emissione di luce di debole intensità nello spettro visibile, da parte di semi in germinazione. E argomento del presente lavoro discutere alcune accurate misure di intensità luminosa condotte su semi germinanti e su loro estratti acquosi in varie condizioni di età della plantule da 1 a 20 giorni. Viene anche studiata la dipendenza dell'emissione luminosa dal pH della soluzione e viene indicata la distribuzione spettrale della luce emessa.     

Loss of bioluminescence inAnomalops katoptron due to starvation

Summary After 3 weeks of starvation the bacterial light-organs of the bioluminescent shallow-water fishAnomalops katoptron cease to produce light. Because of a reduction of the number of symbionts in the cells of the light organ, it is concluded that the fish supplies its luminescent bacteria with nutrients out of its own metabolism. As a result of starving the fish, the luminescent bacteria decrease in number and finally cease to emit light.Supported by a Queen Elizabeth II Fellowship in Marine Science, and carried out during the 1975 Alpha Helix South East Asia Bioluminescence Expedition.     

Mapping and quantification of biomolecules in tumor biopsies using bioluminescence

Quantitative bioluminescence and single-photon imaging have been applied for mapping concentration distributions of metabolites, such as adenosine triphosphate (ATP), glucose and lactate, in biopsies of cervical cancers in patients. Biopsies were taken before a conventional radiation treatment, and a number of clinically relevant data, such as local tumor control, patient survival, metastatic spread and so forth, were documented. There was no correlation between staging or grading and any of the metabolic parameters measured. Local correlations between ATP, glucose and lactate on a pixel-to-pixel basis were generally positive, with respective Spearman's correlation coefficients less in patients without clinically documented metastasis compared with those with metastatic spread. Lactate concentrations were significantly higher and scattered over a wider range in tumors with metastatic spread in comparison to malignancies in patients without metastasis. Thus, high local lactate levels of 20 mole/g appear to be associated with a high risk of metastasis, at least in human cervical tumors.     

Resilin in the lens cuticle of the firefly,Photinus pyralis Linnaeus

Résumé C'est la première fois que de la résiline a été identifiée dans la cornée dePhotinus pyralis Linnaeus.

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I am thankful to Dr.Horace O.Lund for providing facilities in his Department to carry out this work.     

The role of hsp70 in protection and repair of luciferase activity in vivo; experimental data and mathematical modelling

The stably transfected rat cell line HR24 expressing high levels of the inducible human hsp70 and its parental cell line Rat-1 were used for in vivo studies to analyse the role of hsp70 during thermal protein denaturation and the subsequent renaturation. In order to monitor denaturation and renaturation of a cellular protein in vivo, both cell lines were transiently transfected with firefly luciferase (Luc). The continuous monitoring of Luc activity during and after heat stress allowed a detailed analysis of the inactivation and reactivation kinetics in cells grown in monolayers. The aim of these studies was to distinguish a protective effect of increased hsp70 levels during heat shock-induced protein inactivation from a stimulation of reactivation. In this paper we show that in cells that are stably transfected with hsp70, thermal Luc inactivation decreased, and subsequent reactivation yielded higher activity levels, compared with the parental cells. The difference in early inactivation kinetics observed in the two cell lines suggests an immediate effect of the presence of an extra amount of hsp70 on enzyme inactivation. Using different mathematical models, the heat-induced inactivation and reactivation kinetics was compared with simulations of denaturation and renaturation. It is concluded that the model in which it is assumed that hsp70 is able to interact with partially denatured proteins, which did not yet lose their enzymatic activity, most optimally explains the experimental observations. Received 2 December 1998; received after revision 19 February 1999; accepted 18 March 1999     

Molecular diagnosis of parasites

Summary New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene clonign and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.