General anaesthetics and bacterial luminescence II. The effect of diethyl ether on the in vivo light emission of Vibrio fischeri

1976 ◽  
Vol 193 (1111) ◽  
pp. 173-190 ◽  
Keyword(s):  
Diethyl Ether ◽  
Light Emission ◽  
Vibrio Fischeri ◽  
General Anaesthetic ◽  
Bacterial Luminescence

The factors which determine the response of the in vitro luminescent reaction of Vibrio fischeri ,to the general anaesthetic diethyl ether, have been determined. The investigations show that, as was indicated by a study of the in vivo reaction, the levels of substrates available to the enzyme luciferase modify its response to ether. The results indicate that ether inhibits the binding of the aldehyde factor necessary for luminescence. There is evidence that it also acts as a second site where its presence appears to stimulate the binding of reduced flavin to the enzyme.

General anaesthetics and bacterial luminescence I. The effect of diethyl ether on the in vivo light emission of Vibrio fischeri

1976 ◽  
Vol 193 (1111) ◽  
pp. 159-171 ◽  
Keyword(s):  
Diethyl Ether ◽  
Light Emission ◽  
Vibrio Fischeri ◽  
Early Period ◽  
Potassium Cyanide ◽  
Growth Cycle ◽  
Lag Phase ◽  
General Anaesthetic ◽  
Bacterial Luminescence

The factors which determine the sensitivity of bacterial luminescence to inhibition by the general anaesthetic, diethyl ether, have been investigated. The in vivo luminescent reaction of Vibrio fischeri displays a change in sensitivity to this agent during the bacterial growth cycle. This variation is particularly marked during the lag phase of growth and detailed investigations of the effect of potassium cyanide and n -decanal on the potency of ether during this early period are described. The results suggest that fluctuations in the substrate levels available to the light-producing enzyme are responsible for the variation in sensitivity to ether.

scholarly journals INFLUENCE OF ANESTHESIA ON EXPERIMENTAL NEUROTROPIC VIRUS INFECTIONS

1946 ◽  
Vol 84 (4) ◽  
pp. 277-292 ◽  
Author(s):  
S. Edward Sulkin ◽  
Christine Zarafonetis ◽  
Andres Goth
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Diethyl Ether ◽  
Cervical Region ◽  
Virus Infections ◽  
Ether Anesthesia ◽  
Neurotropic Virus ◽  
Experimental Infections

Anesthesia with diethyl ether significantly alters the course and outcome of experimental infections with the equine encephalomyelitis virus (Eastern or Western type) or with the St. Louis encephalitis virus. No comparable effect is observed in experimental infections produced with rabies or poliomyelitis (Lansing) viruses. The neurotropic virus infections altered by ether anesthesia are those caused by viruses which are destroyed in vitro by this anesthetic, and those infections not affected by ether anesthesia are caused by viruses which apparently are not destroyed by ether in vitro. Another striking difference between these two groups of viruses is their pathogenesis in the animal host; those which are inhibited in vivo by ether anesthesia tend to infect cells of the cortex, basal ganglia, and only occasionally the cervical region of the cord. On the other hand, those which are not inhibited in vivo by ether anesthesia tend to involve cells of the lower central nervous system and in the case of rabies, peripheral nerves. This difference is of considerable importance in view of the fact that anesthetics affect cells of the lower central nervous system only in very high concentrations. It is obvious from the complexity of the problem that no clear-cut statement can be made at this point as to the mechanism of the observed effect of ether anesthesia in reducing the mortality rate in certain of the experimental neurotropic virus infections. Important possibilities include a direct specific effect of diethyl ether upon the virus and a less direct effect of the anesthetic upon the virus through its alteration of the metabolism of the host cell.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

scholarly journals LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

2007 ◽  
Vol 190 (5) ◽  
pp. 1531-1538 ◽  
Author(s):  
Sarayut Nijvipakul ◽  
Janewit Wongratana ◽  
Chutintorn Suadee ◽  
Barrie Entsch ◽  
David P. Ballou ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Biochemical Properties ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Knockout Mutant ◽  
Prosthetic Group ◽  
Bacterial Luminescence ◽  
Luminous Bacteria

ABSTRACT The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and k cat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s−1, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH− for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH− for the luminescence reaction in vivo.

scholarly journals PDTM-23. DELTA-24-RGD ONCOLYTIC ADENOVIRUS MEDIATES ANTI-TUMOR EFFECT IN LOCALIZED AND DISSEMINATED AT/RT MURINE MODELS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi192
Author(s):  
Marc Garcia-Moure ◽  
Marisol González Huarriz ◽  
Virginia Laspidea ◽  
Lucía Marrodán ◽  
Candelaria Gomez-Manzano ◽  
...  
Keyword(s):  
Overall Survival ◽  
Brain Tumors ◽  
Phase I ◽  
Light Emission ◽  
Pediatric Brain Tumors ◽  
Oncolytic Adenovirus ◽  
Intraventricular Injection ◽  
Pediatric Brain

Abstract Atypical teratoid/rhabdoid tumors (AT/RTs) are rare pediatric brain tumors affecting mainly infants and young children. However, AT/RTs encompass almost 10% of death caused by pediatric brain tumors, and the 2-year overall survival for these children remains below 20%. For this reason, AT/RT ranks among the deadliest pediatric brain tumors. Therefore, it is clear we need to find out new therapeutic options for these children. Delta-24-RGD oncolytic adenovirus has already demonstrated its efficacy in Phase I/II clinical trials in adult patients affected by high grade gliomas with no evidence of severe side effects. Of interest for pediatric brain tumors, the safety of Delta-24-RGD is has been demonstrated in an ongoing Phase I clinical trial for the treatment of DIPGs (NCT03178032). For these reasons, we propose to evaluate the anti-tumor effect of Delta-24-RGD in preclinical models of AT/RT. In vitro, the virus was able to infect and replicate in three different cell culture models of AT/RT, inducing a dose-dependent cytotoxic effect that results in IC50 values below 1 PFU/cell. In vivo, intratumor administration of the virus in mice bearing orthotopic localized AT/RT (supratentorial and infratentorial) extended significantly the survival the animals, leading to up to 20% of long-term survivors. We have also generated models of disseminated disease through intraventricular injection of the tumor cells, thus mimicking the lesions found in patients. AT/RT cell lines were transduced with a luc-expressing lentivirus in order to facilitate the follow up of these tumors. In disseminated AT/RT models, light emission reveals reduction of tumor growth in Delta-24-RGD treated animals in comparison to those mock treated, thus obtaining an increased overall survival. In conclusion, these results demonstrate that Delta-24-RGD could be a feasible therapeutic choice for patients affected by AT/RT.

The escal light gland of the deep-sea anglerfish Haplophryne mollis (Pisces: Ceratioidei) with observations on luminescence control

1994 ◽  
Vol 74 (4) ◽  
pp. 747-763 ◽  
Author(s):  
Peter J. Herring ◽  
Ole Munk
Keyword(s):  
Deep Sea ◽  
Light Emission ◽  
Light And Electron Microscopy ◽  
Symbiotic Bacteria ◽  
Uniform Structure ◽  
Basal Cells ◽  
Bacterial Luciferase ◽  
The Right

The escal light gland of three different-sized specimens of the deep-sea anglerfish Haplophryne mollis (family Linophrynidae) has been examined by light and electron microscopy. The light gland has a central cavity, with diverging branched ducts which ramify into numerous tightly-packed radial tubules. In the two largest specimens all glandular lumina contain symbiotic bacteria. Except for a thin-walled part of the typical radiating tubules, the epithelial walls of the light gland are of a uniform structure, consisting of flattened basal cells, situated next to the basal lamina, and tall cells extending to the lumen.In the smallest specimen examined the various parts of the light gland were not fully differentiated and only a very few symbiotic bacteria were present; its glandular epithelium differed from that of the two larger specimens by containing many goblet cells, the secretion of which may be important for the initial establishment of the right strain of symbiotic bacteriaObservations on the luminescence of live specimens have shown that the light emission can be rapidly modulated from within the esca. The in vivo flash kinetics are considerably slower than those of Dolopichthys longicornis, but similar to those of both the caruncle exudate of Ceratias holboelli and in vitro anglerfish bacterial luciferase.

scholarly journals Involvement of the RNA Polymerase α-Subunit C-Terminal Domain in LuxR-Dependent Activation of the Vibrio fischeri Luminescence Genes

1999 ◽  
Vol 181 (15) ◽  
pp. 4704-4707 ◽  
Author(s):  
Ann M. Stevens ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
E. P. Greenberg
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Vibrio Fischeri ◽  
In Vivo Studies ◽  
Wild Type ◽  
Α Subunit ◽  
Subunit C ◽  
Terminal Domain

ABSTRACT LuxR is a ς70 RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the Vibrio fischeri lux operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the α-subunit C-terminal domain (αCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRΔN) was used to study the activation of transcription from theluxI promoter in vitro. Initiation of luxoperon transcription was observed in the presence of LuxRΔN and wild-type RNAP but not in the presence of LuxRΔN and RNAPs with truncated αCTDs. We also studied the in vivo role of the RNAP αCTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRΔN. These in vivo studies indicated that both LuxR and LuxRΔN require the RNAP αCTD for activity. The results of DNase I protection studies showed that LuxRΔN-RNAP complexes can bind and protect the luxIpromoter, but with less efficacy when the αCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the lux operon involves the RNAP αCTD and suggest that αCTD-LuxR interactions may play a role in recruitment of RNAP to theluxI promoter.

scholarly journals Bioluminescence as a tool in molecular biology

2017 ◽  
Vol 71 (0) ◽  
pp. 0-0
Author(s):  
Katarzyna Pajor ◽  
Daniel Sypniewski ◽  
Ilona Bednarek
Keyword(s):  
Molecular Biology ◽  
Protein Interactions ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Protein Protein Interactions ◽  
Photinus Pyralis ◽  
Bacterial Bioluminescence ◽  
Lux Operon

Bioluminescence has been studied for many years by scientists. There are numerous mechanisms of that phenomenon; among them bacterial bioluminescence is the most frequently found in nature. This type of bioluminescence is determined by the appearance of lux operon, which encodes all elements necessary to produce light emission and it does not require any additional substrates supply. Another commonly found example of bioluminescence mechanism is performed by Photinus pyralis. Luciferase of P. pyralis named FLuc requires D-luciferin as a substrate. Bioluminescence is also characteristic for many deep-sea organisms. Most of them are based on oxidation reaction of coelenterazine to coelenteramide mediated by RLuc or GLuc luciferases. Due to the variety of bioluminescence mechanisms in nature, it has become possible to apply them in many sensitive methods that can be used in molecular biology and medicine. The most significant application of bioluminescence is BLI (bioluminescence imaging). This method is cheap and nontoxic which allows both in vitro and in vivo imaging. BLI applications include, e.g. protein-protein interactions, stem cells labeling, tracking of viral, bacterial, fungal and parasitical infections, and carcinogenesis analyses. Bioluminescence has also been used in the creation of modified cell systems capable of light emission in response to certain analytes and thus very sensitive biosensors have been generated. Other important areas of bioluminescence application are immunoassays, ATP assays, and BART analysis (bioluminescent assay in Real-Time) – a very sensitive technique which allows scientists to estimate nucleic acids amplification.

scholarly journals Evaluation of Ralstonia solanacearum Infection Dynamics in Resistant and Susceptible Pepper Lines Using Bioluminescence Imaging

Plant Disease ◽  
2017 ◽  
Vol 101 (2) ◽  
pp. 272-278 ◽  
Author(s):  
Heshan Du ◽  
Bin Chen ◽  
Xiaofen Zhang ◽  
Fenglan Zhang ◽  
Sally A. Miller ◽  
...  
Keyword(s):  
Real Time ◽  
Ralstonia Solanacearum ◽  
Light Emission ◽  
Nondestructive Method ◽  
Resistant Line ◽  
In Planta ◽  
Susceptible Line ◽  
Infection Dynamics

Bacterial wilt, incited by Ralstonia solanacearum, is a major disease affecting pepper (Capsicum annuum) production worldwide. The most effective management tactic is the deployment of wilt-resistant varieties. However, the lack of a nondestructive method to measure invasiveness and spatio-temporal distribution of R. solanacearum, a vascular pathogen, in planta limits better understanding of pepper resistance and plant-pathogen interactions. We evaluated the resistance of 100 pepper lines using R. solanacearum strain Rs-SY1 (phylotype I, isolated from a sweet pepper in South China). Based on the disease severity index (DSI) values, the elite inbred line BVRC 1 and the small-fruited accessions PI 640435 and PI 640444 were identified as resistant (DSI: 1.2, 1.8, and 1.9 out of 4.0, respectively). In order to evaluate bacterial infection dynamics in planta in real time, we generated seven bioluminescent R. solanacearum strains (BL-Rs1 to BL-Rs7) using vector pXX3 carrying luxCDABE genes, and selected BL-Rs7 for inoculation due to its similarity with parent strain Rs-SY1 in morphology, pathogenicity, and highest light emission in vitro. Luminescence intensity was strongly correlated to bacterial population in planta (R2 = 0.88). The utility of the bioluminescence assay was validated by comparing R. solanacearum infection dynamics in real-time in vivo between resistant line BVRC 1 and susceptible line BVRC 25. The distribution and multiplication of BL-Rs7 strain in resistant line BVRC 1 was conspicuously limited in plants inoculated in either roots or stem compared with susceptible line BVRC 25. These results suggest that pepper line BVRC 1 may resist colonization by interfering with R. solanacearum multiplication in the roots and stem.

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