A report on methods to reduce,refine and replace animal testing in industrial toxicology laboratories

The Committee to Promote Principles of Reduction, Refinement and Replacement of Animal Testing in Industrial Toxicology Laboratories was established in 1987 to work toward industrywide improvements in laboratory animal testing methods. The committee's goals are to gather information about effective nonanimal testing techniques and other methods of conserving and improving the care of laboratory animals, to work toward the systematic validation of nonanimal alternatives, and to disseminate useful information about progressive programs and policies throughout the industrial toxicology community. This is the first in a continuing series of reports the committee plans to produce as part of an ongoing program to promote communication among industrial toxicologists about successful methods of reducing, refining and replacing animal testing. Here are some of the report's major findings: (1) Animal care and use committees charged with the oversight of laboratory animal use are a universal practice at the companies surveyed. (2) Significant reductions in the number of animals used for acute toxicity testing have taken place at all the companies during the last 5- to 10-year period. (3) Structure-activity relationships (predicting a test compound's properties based on the known properties of familiar chemicals with similar structures) are widely used to minimize, but not replace, the use of animals. (4) Tissue and organ culture systems are being used with increasing frequency for screening and mechanistic studies, but are not completely replacing animal evaluations as a final step. (5) There is a pressing need for the systematic and scientifically sound validation of nonanimal alternative techniques to reduce the use of animals in toxicology testing while satisfying requirements for the protection of public safety.     

Swiveling and decatenation of replicating simian virus 40 genomes in vivo.

We have found that type II topoisomerase inhibitors have two effects on replicating simian virus 40 genomes in vivo: production of catenated dimers and slowed replication of the last 5% of the genome. This suggests that type II topoisomerase simultaneously decatenates and facilitates replication fork movement at this stage of DNA replication. On the basis of this observation, a detailed model is proposed for the roles of topoisomerases I and II in simian virus 40 DNA replication.     

Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (相似文献   

Invertebrate communities associated with a native (Vallisneria americana) and an alien (Trapa natans) macrophyte in a large river

1. We used a corer and a Downing box sampler to sample macroinvertebrates living on and beneath the introduced Trapa natans and the native Vallisneria americana in the freshwater tidal Hudson River, New York. 2. Densities of macroinvertebrates were higher in Trapa than in Vallisneria, and higher in the interior of plant beds than at their edges. These effects were largely a result of high plant biomass in Trapa beds and in bed interiors (the plants have similar surface area per unit mass). 3. The composition of both epiphytic and benthic macroinvertebrates differed distinctly between Trapa and Vallisneria, and also seasonally. 4. These compositional differences were not easily interpretable as rising from possible differences in oxygen concentrations, fish predation, or water circulation in the two macrophytes. 5. Sida crystallina (Cladocera) collected from Trapa contained more haemoglobin than those collected from Vallisneria. 6. The replacement of Vallisneria by Trapa in the Hudson probably increased system‐wide biodiversity and food for fish, although macroinvertebrates in Trapa beds may not be readily available to fish because of low oxygen concentration there.     

Total antioxidant capacity – a novel early bio-chemical marker of oxidative stress in HIV infected individuals

Background  

Oxidative stress induced by the production of reactive oxygen species may play a critical role in the stimulation of HIV replication and the development of immunodeficiency. This study was conducted as there are limited and inconclusive studies on the significance of a novel early marker of oxidative stress which can reflect the total antioxidant capacity in HIV patients,     

Molecular determinants of survival motor neuron (SMN) protein cleavage by the calcium-activated protease, calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Reduced Sulfur in Ashes and Slags from the Gasification of Coals: Availability for Chemical and Microbial Oxidation

This study was initiated to determine if reduced sulfur contained in coal gasifier ash and slag was available for microbial and chemical oxidation because eventual large-quantity landfill disposal of these solid wastes is expected. Continuous application of distilled water to a column containing a high-sulfur-content (4% [wt/wt]) gasifier slag yielded leachates with high sulfate levels (1,300 mg of sulfate liter⁻¹) and low pH values (4.2). At the end of the experiment, a three-tube most-probable-number analysis indicated that the waste contained 1.3 × 10⁷ thiosulfate-oxidizing bacteria per g. Slag samples obtained aseptically from the column produced sulfate under both abiotic and biotic conditions when incubated in a mineral nutrient solution. Both microbial and chemical sulfate syntheses were greatly stimulated by the addition of thiosulfate to the slag-mineral nutrient solution. These results led to a test of microbial versus chemical sulfur oxidation in ashes and slags from five gasification processes. Sulfate production was measured in sterile (autoclaved) and nonsterile suspensions of the solid wastes in a mineral nutrient solution. These ashes and slags varied in sulfur content from 0.3 to 4.0% (wt/wt). Four of these wastes demonstrated both chemical (2.0 to 27 μg of sulfate g⁻¹ day⁻¹) and microbial (3.1 to 114 μg of sulfate g⁻¹ day⁻¹) sulfur oxidation. Obvious relationships between sulfur oxidation rate and either sulfur content or particle size distribution of the wastes were not immediately evident. We conclude that the sulfur contained in all but one waste is available for oxidation to sulfuric acid and that microorganisms play a partial role in this process.