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Biomedical subjects

L Masson

Publications and source records attributed to L Masson.

At least 55 records · Page 3Linked to original sources
PubMed1993

Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains.

Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates. Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis. This method has been used to distinguish commercial products containing B. thuringiensis serovar kurstaki (3a3b). When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars. Differentiation from the closely related species Bacillus cereus is also readily achieved. This technique should prove to be a powerful tool for identification and discrimination of individual B. thuringiensis strains.

Bacillus cereus↗
PubMed1993

Construction and application of chromosomally integrated lac-lux gene markers to monitor the fate of a 2,4-dichlorophenoxyacetic acid-degrading bacterium in contaminated soils.

A reporter gene system, containing luxAB and lacZY, was constructed and integrated, using Tn7 transposition, into the chromosome of a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading soil bacterium, Pseudomonas cepacia (BRI6001), to monitor its fate when introduced into soil microcosms. The genes were stably maintained in the modified strain of BRI6001, BRI6001L, for more than 300 generations in the absence of selection pressure, and had no apparent effects on biochemical or physiological properties. BRI6001L was easily and rapidly identified as light-emitting blue colonies on 2,4-D medium containing XGal (5-bromo-4-chloro-indolyl-beta-D-galacto-pyranoside) in the presence of n-decanal. Survival rates of BRI6001L introduced into non-sterile soil microcosms were substrate- and contaminant-dependent. The decrease in population density was lowest in a 2,4-D-amended agricultural soil, and highest in a wood-treatment facility soil contaminated with pentachlorophenol, creosote and heavy metals. A viable cell density as low as 10 cfu g-1 was detected in soil microcosms. The biochemical and growth properties of BRI6001 and BRI6001L, and their behaviour when introduced into soil microcosms indicates that BRI6001L can be used as a reliable model to predict the fate of BRI6001 when used to bioaugment contaminated soil.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Comparative toxicity of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki to defoliating forest Lepidoptera.

Insecticidal activities of sporulated cultures of the HD-1 and NRD-12 strains of Bacillus thuringiensis subsp. kurstaki were compared against four species of defoliating forest lepidopterans in diet-incorporation assays. There was no difference in LC50 between the two strains to larvae of spruce budworm (Choristoneura fumiferana), gypsy moth (Lymantria dispar), eastern hemlock looper (Lambdina fiscellaria fiscellaria), and whitemarked tussock moth (Orgyia leucostigma), whether expressed as total alkaline soluble protein, activated toxin protein, or International Units as determined by bioassay against Trichoplusia ni. Both strains were consistently more toxic than HD-1-S-1980 when compared on the basis of alkali-soluble protein, but not on the basis of activated toxin or International Units. Hybridization of genomic DNA after restriction with HindIII revealed the presence of all three cryIA toxin genes in each of the isolates used in this study, including HD-1-S-1980, which was previously reported to have lost the cryIA(b) gene.

Animals↗
PubMed1992

Specificity of the Pseudomonas aeruginosa PAO1 lipoprotein I gene as a DNA probe and PCR target region within the Pseudomonadaceae.

The lipoprotein I gene (oprI) of Pseudomonas aeruginosa PAO1 was cloned and sequenced. A high degree of homology was found between our cloned PAO1 gene sequence and two published oprI sequences. Specific oligonucleotides were designed to amplify the oprI gene by the polymerase chain reaction (PCR). The potential of either the complete gene sequence or the specific oligonucleotide primers as a tool for rapid strain identification was directly assessed against bacterial colonies by PCR or against purified genomic DNA by Southern blot analysis, using a number of representative strains within the Pseudomonadaceae. The oprI gene was found to be well conserved within RNA group I.

Amino Acid Sequence↗
PubMed1992

Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae.

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.

Animals↗
PubMed1991

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.

Animals↗
PubMed1991

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera.

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD(50)]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD(50) of HD-1 with the FFD(50) expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.

Journal Article↗
PubMed1991

[Active rheumatoid arthritis: effect of dietary supplementation with omega-3 oils. A controlled double-blind trial].

We evaluated the effect of dietary supplementation with eicosapentaenoic acid in 8 patients with active rheumatoid arthritis. An appropriate placebo was given in a randomized double blind fashion to 8 control subjects. After 12 weeks of therapy a significant improvement in prehensile function was detected in patients receiving active treatment, other clinical parameters remaining unchanged. No significant side effects were detected. A larger trial may help define a possible therapeutic role for omega-3 fatty acids in patients with rheumatoid arthritis.

Adult↗
PubMed1990

Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1.

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.

Animals↗
PubMed1990

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12.

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.

Animals↗
PubMed1989

Transformation of Bacillus thuringiensis vegetative cells by electroporation.

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.

Bacillus thuringiensis↗
PubMed1988

Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product.

E protein, the 29 Kd repE gene product, is essential for the replication of the Escherichia coli F plasmid. The repE gene has been cloned and expressed in an inducible ATG-fusion vector, and the protein product has been purified to homogeneity. The purified E protein is present as a dimer in solution and binds specifically to both the 19-bp direct repeats (incB) found within the mini-F origin of replication ori2 and the repE operator DNA. Examination of the amino acid sequence of E protein revealed a consensus sequence for DNA binding.

Amino Acid Sequence↗
PubMed1988

Bacillus thuringiensis insecticidal crystal toxins: gene structure and mode of action.

Thanks to the techniques of recombinant DNA, there is now abundant sequence information on several endotoxin genes of Bacillus thuringiensis. The task of correlating this sequence information with the economically important aspects of the toxins such as insect specificity, LD(50) and speed of kill is now under worldwide investigation. Progress has also been made on understanding the mechanism of action of the toxins and on identifying the parts of the protoxin which are important in toxicity. Taken together, the mechanistic data and the sequence information allow the first attempts at rational design of mutant endotoxin genes and greatly facilitate the transfer of those genes to other organisms such as plants. More information is still needed, however, as to the nature of the binding site of the toxin and on the three-dimensional structure of the activated toxins.

Journal Article↗
PubMed1986

Mechanism of autonomous control of the Escherichia coli F plasmid: different complexes of the initiator/repressor protein are bound to its operator and to an F plasmid replication origin.

E protein, the 29 kd product of the F plasmid repE gene, plays both positive and negative roles in the autoregulation of F replication. We have cloned and expressed the repE gene in an inducible ATG-fusion vector and have detected specific binding of E protein to the repE operator and to four 19-base pair direct repeats (incB) within the F plasmid replication origin ori2. Binding of E protein at the repE operator occurs with higher affinity than at ori2(incB) and gives almost complete protection to at least 30 base pairs, whereas binding of E protein to the direct repeats in the ori2 region shows an alternating pattern of enhanced and reduced sensitivity to DNAase cleavage consistent with a protein-induced folding of the DNA. These results provide direct biochemical support for a model of F plasmid replication in which the E protein serves both as an initiator of replication and as an autorepressor of its own synthesis.

Base Sequence↗