摘要:

AbstractDuring the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–‐the L‐929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC50values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and ir

ISSN:0884-3996    

DOI:10.1002/bio.1170050202

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractTaking advantage of a specially constructed vector, luciferase LuxA and LuxB subunits were connected in frame to different amino acid linkers to reproduce a series of monomeric luciferase enzymes. A comparison of their activities inE. colicells demonstrated that the length of the linkers positively affected activity. One luciferase fusion gene was expressed in plant cells, and we showed that this gene activity could be monitored directly without destructive sampling.

ISSN:0884-3996    

DOI:10.1002/bio.1170050203

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractLuciferase fromVibrio harveyiis encoded by two adjacent genes,luxAandluxB. The two genes were fused by replacing a segment extending from near the end ofluxAinto the N‐terminal end ofluxBby a synthetic oligonucleotide. The construction removed the TAA stop codon at the end ofluxA, the intervening region of 26 base pairs, and the initial methionine ofluxB. A Smal site was included at the junction between the two genes and an Aatll site was created near the end ofluxAwithout altering its amino acid sequence. InEscherichia colithe fusedluxABgene could be expressed to produce functional luciferase that gave about 20% of the activity in cells without the fusion.An out‐of frame ATG exists close to and preceding the ATG of theluxAgene. This was removed and the entire fused gene bracketed by several restriction enzyme sites.The fusedluxABgene was successfully expressed inSaccharomyces cerevisiaeandDrosophila melanogasterby transferring it to appropriate plasmid vect

ISSN:0884-3996    

DOI:10.1002/bio.1170050204

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractRegulation of expression of bioluminescence from theVibrio fischeri luxregulon inEscherichia coliis a consequence of a unique form of positive feedback superimposed on a poorly definedcis‐acting repression mechanism. Theluxregulon consists of two divergently transcribed operons. The leftward operon contains only a single gene,luxR, which encodes a transcriptional activator protein. The rightward operon containsluxl, which together withluxRand the 218 base pairs separating the two operons comprises the primary regulatory circuit, and the five structural genes,luxC, luxD, luxA, luxBandluxE, which are required for the bioluminescence activity. Transcription ofluxRfrom PLis stimulated by binding of theE. coli crpgene product to the sequence TGTGACAAAAATCCAA upstream of the presumed promoter. Binding of pureE. coliCAP protein in a cAMP‐ dependent reaction to theV. fischeri luxregulatory region has been demonstrated byin vitrofootprinting. Theluxlgene product is an enzyme which catalyses a condensation reaction of cytoplasmic substrates to yield the autoinducer,N‐(3‐oxo‐hexanoyl) homoserine lactone. Accumulation of autoinducer, which is freely diffusible, results in formation of a complex with LuxR. The complex binds to the sequence ACCTGTAGGATCGTACAGGT upstream of PRto stimulate transcription of the rightward operon. Increased transcription fromPRshould yield increased levels of Luxl and higher levels of autoinducer which would further activate LuxR. The LuxR binding site is also a LexA binding site, as demonstrated byin vitrofootprinting. Basal transcription from both PLand PRis repressed by sequences within theluxRcoding region. Hence there appear to be at least two effects resulting from the interaction between LuxR: autoinducer and the control region DNA. One effect is to relieve the repression afforded by the sequences withinluxRand the second is to stimulate transcription from PR. Recent analysis of the rightward promotor by site‐directed mutagenesis has suggested a different location for PRthan that which was implicated in earlier studies. Our results suggest that the −35 sequence is located at a position which overlaps the 3′ edge of the LuxR binding site b

ISSN:0884-3996    

DOI:10.1002/bio.1170050205

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractLuminescence assays are generally based on measurements of light intensity alone. Inclusion of colour as an additional parameter of the assay could increase the information content. Colour variation in luminescence is particularly prevalent among beetle luciferases. To study the relationship between enzyme structure and colour, luciferases from a Jamalcan click beetle were examined as a model system. These luciferases emit light ranging from green to orange, though their amino acid sequences differ by less than 5%. Through mutation of their respective cDNA clones, the amino acids responsible for the colour variation were identified. These specific amino acids are few, and they act upon colour independently with respect to the enzyme structure. Analysis of their effects indicates that the potential for colour variation among beetle luciferases is greater than is evident among the click beetle luciferase. Because of the subtle changes of enzyme structure that effect colour, luciferases that emit different colours may be useful as paired genetic reporters. They should interact equivalently with the intracellular environment of a host, but could be distinguished by colour in their assay. Such paired reporters could be used to observed simultaneous events, or to provide internal control for luminescence measurements.

ISSN:0884-3996    

DOI:10.1002/bio.1170050206

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractThe detection of specific bacterial pathogens, indicator microorganisms and antimicrobial substances, and the recovery of microorganisms from sub‐lethal injury, are all aspects of importance to industry which are currently being targeted usingin vivobioluminescence. In all instances, a key requirement for the application of bioluminescence is the establishment of a strict correlation betweenin vivobioluminescence and cell viability, as determined by colony counting on agar plates. Comparative studies for biocides (phenol, chlorhexidine diacetate, phenol thioether), for a virucide (hypochlorite) and for cellular recovery ofS. typhimuriumfrom sub‐lethal injury, all indicate that such a correlation is valid. Furthermore, real‐time measurements ofin vivobioluminescence reveal a major population of bacterial cells that retain functional intracellular biochemistry, but are defective in their ability to replicate post of freeze i

ISSN:0884-3996    

DOI:10.1002/bio.1170050207

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractQuantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooperet al., 1990). A brief review of low‐light‐level imaging is presented, with particular reference to charge‐coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photoncounting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular highsensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguezet al., 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non‐infected cells. The rapid detection of luciferase‐expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles.This direct imaging technique is simple and versatile. It offers a rapid, non‐invasive method for the sensitive detection of luciferase activity in single, luciferase‐expressing cells. One can envisage the use of luciferase as a sensitive and convenient co‐selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and c

ISSN:0884-3996    

DOI:10.1002/bio.1170050208

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractTheLucgene from the fireflyPhotinus pyralishas been isolated by cloning it in pcDV1 PL plasmid primer and Honjo linker and the Phot gene isolated fromAequorea victoriausing the polymerase chain reaction. A method has been established using SP6 RNA polymerase for transcribing and translating bioluminescent genesin vitro.It should now be possible to engineer these genes to measure intracellular ATP and the covalent modification of proteins in single, live cells, providing unique insights into the molecular basis of disease.

ISSN:0884-3996    

DOI:10.1002/bio.1170050209

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

摘要:

AbstractThis is the first of a series of special compilations of references devoted to a particular topic in luminescence. References are numbered sequentially, except when a reference has appeared in a previous Bioluminescence and Chemiluminescence Literature section in which case it retains the original number.

ISSN:0884-3996    

DOI:10.1002/bio.1170050210

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY

ISSN:0884-3996    

DOI:10.1002/bio.1170050211

出版商:John Wiley&Sons, Ltd.    

年代:1990

数据来源: WILEY