Frequency of the mutant MDR1 allele associated with multidrug sensitivity in a sample of collies from France.

A study was performed to determine the frequency of the mutant MDR1 allele associated with ivermectin sensitivity in a sample of collies living in France. Buccal swab samples were collected from approximately 83 collies for determination of MDR1 genotype. DNA was extracted and the polymerase chain reaction was performed to amplify a 148 bp (wildtype MDR1 genotype) or 144 bp (mutant MDR1 genotype) amplicon containing the MDR1 mutation. Sequence analysis was performed to determine the genotype of each dog. Adequate quantities of DNA for unequivocal genotyping were obtained from only 25 of 83 swabs. Twenty percent (5/25) of the collies studied were homozygous for the normal allele (normal), 32% (8/25) were heterozygous (carrier), and 48% (12/25) were homozygous for the mutant allele (affected). The results of this study indicate that a high percentage of collies presenting to veterinarians in France harbor the MDR1 mutation, thus impacting some therapeutic decisions.

Dexamethasone treatment of a canine, but not human, tumour cell line increases chemoresistance independent of P-glycoprotein and multidrug resistance-related protein expression.

Glucocorticoids are often used in veterinary cancer patients because of their anti-inflammatory actions, appetite-stimulating effects, ability to decrease nausea and vomiting associated with some chemotherapy agents, and, in some instances, for their cytotoxic actions on susceptible tumour cells. Veterinary oncologists may not consider the possibility that the use of glucocorticoids may adversely affect response to chemotherapy. There is evidence that glucocorticoids can up-regulate the expression of multidrug resistance genes in some tissues. Whether or not glucocorticoid-induced expression of multidrug resistance proteins occurs in tumour cells is not presently known. The purpose of this study was to determine if dexamethasone induces P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1) in tumour cell lines. A canine osteosarcoma cell line (OS2.4) and a human myeloid leukaemia cell line 60 (HL60) were treated in culture with dexamethasone. The presence of a glucocorticoid receptor was confirmed in both cell lines by reverse-transcriptase polymerase chain reaction. Western blots for P-gp and MRP1 expression were performed on vehicle-treated and dexamethasone-treated cells. Sensitivity towards several chemotherapeutic drugs (cisplatin (cis-diamminedichloroplatinum), doxorubicin, methotrexate and vincristine) was determined by 3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. While dexamethasone treatment of OS2.4 cells increased the resistance to cisplatin and methotrexate, an increase in P-gp or MRP1 expression was not observed. Dexamethasone-treated HL60 cells did not develop chemoresistance and did not show increased expression of P-gp or MRP1.

Sequence and structural analysis of the presumed downstream promoter of the canine mdr1 gene.

The product of the canine mdr1 gene, P-glycoprotein (P-gp), plays an important role in chemotherapeutic drug resistance of several canine tumours. Increased expression of P-gp by tumour cells is associated with the multidrug-resistant phenotype. Because of its importance in cancer chemotherapy, a great deal is known about the regulation of mdr1 gene expression in human cancer patients and rodent cancer models. In contrast, there is no information regarding the regulation of P-gp expression in dogs. Initial information regarding the regulation of mdr1 gene expression can be gained by evaluating the mdr1 promoter. The downstream promoter of the canine mdr1 gene was sequenced. Several regulatory elements were identified, including an AP-1 site, AP-2 site and SP-1 site. The presumed canine mdr1 promoter was similar to that of other species; however, low overall sequence homology may suggest that aspects of P-gp regulation are distinctive in dogs.

Ivermectin sensitivity in collies is associated with a deletion mutation of the mdr1 gene.

A subpopulation of collie dogs is extremely sensitive to neurotoxicity induced by ivermectin. The aim of this study was to determine the mechanistic basis for this phenomenon. The multi-drug-resistance gene (mdr1) encodes a large transmembrane protein, P-glycoprotein (P-gp), that is an integral part of the blood-brain barrier. P-gp functions as a drug-transport pump at the blood-brain barrier, transporting a variety of drugs from the brain back into the blood. Since ivermectin is a substrate for P-gp, we hypothesized that ivermectin-sensitive collies had altered mdr1 expression compared with unaffected collies. We report a deletion mutation of the mdr1 gene that is associated with ivermectin sensitivity. The 4-bp deletion results in a frame shift, generating several stop codons that prematurely terminate P-gp synthesis. Dogs that are homozygous for the deletion mutation display the ivermectin-sensitive phenotype, while those that are homozygous normal or heterozygous do not display increased sensitivity to ivermectin.

Effects of a lignin peroxidase-expressing recombinant, Streptomyces lividans TK23.1, on biogeochemical cycling and the numbers and activities of microorganisms in soil.

A recombinant actinomycete, Streptomyces lividans TK23.1, expressing a pIJ702-encoded extracellular lignin peroxidase gene cloned from the chromosome of Streptomyces viridosporus T7A, was released into soil in flask- and microcosm-scale studies to determine its effects on humification and elemental cycling and on the numbers, types, and activities of microorganisms native to the soil. Strain TK23.1 had been shown previously to transiently increase the rate of organic carbon mineralization in soil via an effect that was recombinant specific and particularly significant in nonsterile soils already possessing an active microflora. The results of this study confirmed the previous findings and showed that additional effects were measurable upon release of the recombinant strain TK23.1 into unamended soil and into soil amended with lignocellulose. In addition to a transient enhancement of carbon mineralization, the recombinant affected soil pH, the rate of incorporation of carbon into soil humus fractions, nitrogen cycling, the relative populations of some microbial groups, and also certain soil enzyme activities. Whereas the survival or persistence in soil of the recombinant TK23.1 strain and that of its parent, TK23, were similar, the observed effects on microbial numbers, types, and activities were recombinant specific and did not occur when the parental strain was released into soil. All of the measured effects were transient, generally lasting for only a few days. While the effects were statistically significant, their ecological significance appears to be minimal. This is the first report showing that a recombinant actinomycete can affect the microbial ecology of soil in ways that can be readily monitored by using a battery of microbiological, enzymological, and chemical assays.

Field calibration of soil-core microcosms: Fate of a genetically altered rhizobacterium.

Microcosms containing intact soil-cores are a potential tool for assessing the risks of the release of genetically engineered microorganisms (GEMs) to the environment. Before microcosms become a standard assessment tool, however, they must first be calibrated to ensure that they adequately simulate key parameters in the field. Four systems were compared: intact soil-core microcosms located in the laboratory at ambient temperature and in a growth chamber with temperature fluctuations that simulated average conditions in the field, field lysimeters, and field plots. These four systems were inoculated with rifampicin-resistantPseudomonas sp. and planted to winter wheat. Populations of thePseudomonas sp. in soil decreased more rapidly at ambient temperature, but population size at the three-leaf stage of wheat growth was the same in all four systems. Populations of thePseudomonas sp. on the rhizoplane of wheat were the same at the three-leaf stage in all four systems, and colonization with depth at the final boot stage-sampling was also similar. In general, microcosms incubated at ambient temperature in the laboratory or in the growth chamber were similar to those in the field with respect to survival of and colonization of the rhizoplane by the introducedPseudomonas sp.

Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons.

Microcosms containing intact soil-cores are a potential biotechnology risk assessment tool for assessing the ecological effects of genetically engineered microorganisms before they are released to the field; however, microcosms must first be calibrated to ensure that they adequately simulate key field parameters. Soil-core microcosms were compared with the field in terms of ecological response to the introduction of a large inoculum of a rifampicin-resistant rhizobacterium,Pseudomonas sp. RC1. RC1 was inoculated into intact soil-core microcosms incubated in the laboratory at ambient temperature (22°C) and in a growth chamber with temperature fluctuations that mimicked a verage field values, as well as into field lysimeters and plots. The effect of the introduced bacterium on ecosystem structure, including wheat rhizoplane populations of total and fluorescent pseudomonads, total heterotrophic bacteria, and the diversity of total heterotrophic bacteria, was determined. Fluorescent pseudomonads were present on the rhizoplane in significantly lower numbers in soil inoculated with RC1, in both microcosms and the field. Conditions for microbial growth appeared to be most favorable in the growth chamber microcosm, as evidenced by higher populations of heterotrophs and a greater species diversity on the rhizoplane at the three-leaf stage of wheat growth. Ecosystem functional parameters, as determined by soil dehydrogenase activity, plant biomass production, and(15)N-fertilizer uptake by wheat, were different in the four systems. The stimulation of soil dehydrogenase activity by the addition of alfalfa was greater in the microcosms than in the field. In general, growth chamber microcosms, which simulated average field temperatures, were better predictors of field behavior than microcosms incubated continuously at 22°C.

Intact soil-core microcosms for evaluating the fate and ecological impact of the release of genetically engineered microorganisms.

Intact soil-core microcosms were studied to determine their applicability for evaluating the transport, survival, and potential ecosystem effects of genetically engineered microorganisms before they are released into the environment. Soil-core microcosms were planted with wheat and maize seeds and inoculated with Azospirillum lipoferum SpBr17 and SpRG20a Tn5 mutants, respectively. Microcosm leachate, rhizosphere soil, plant endorhizosphere, insects, and xylem exudate were sampled for A. lipoferum Tn5 mutant populations. A. lipoferum Tn5 populations, determined by most-probable-number technique-DNA hybridization, varied from below detection to 10 g of dry root in the rhizosphere, with smaller populations detected in the endorhizosphere. Intact soil-core microcosms were found to maintain some of the complexities of the natural ecosystem and should be particularly useful for initial evaluations of the fate of plant-associated genetically engineered bacteria.