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.     

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,     

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.     

Bionomics of invertebrates within an extensive Potamogeton pectinatus bed of a California marsh

Fourteen samples of sago pondweed (Potamogeton pectinatus L.) and associated invertebrates were collected every two weeks over a single season of plant growth in a large monospecific pondweed-bed located in Coyote Hills Marsh (Alameda Co., California, USA), using pull-up samplers that collect plants, epiphytic macroinvertebrates, and microcrustaceans throughout the water column. The macro-invertebrate fauna was dominated by insects, primarily chironomids. Semi-aquatic neustonic taxa, including an aphid and a springtail, were common; this is in contrast with most aquatic plant-invertebrate studies, in which neustonic insects are seldom collected because of sampling bias. Over the entire season, P. pectinatus biomass and the densities of four insect taxa (Anopheles spp. mosquitoes, Hydrellia sp. brineflies, Ademon sp. parasitic wasps, and coenagrionid damselflies) were significantly correlated. These correlations resulted from both similar overall phenologies of the plant and each of the insect taxa, and ecological relationships in which P. pectinatus provides either a specialized habitat or food source. macroinvertebrate numbers were highest in mid-summer, when P. pectinatus forms a dense floating canopy; microcrustaceans were more common during plant senescence in early autumn. Individuals of some taxa may be distributed in proportion to plant biomass; this occurred commonly in damselflies, perhaps as a result of territoriality in these nymphs.     

Oral mucosal pellicle. Adsorption and transpeptidation of salivary components to buccal epithelial cells.

The present investigation was carried out to examine the mechanism(s) whereby salivary molecules interact with human buccal epithelial cells. By utilizing antiserum against human parotid saliva, selected salivary components were detected by electrophoretic-transfer analysis of 1.5% SDS extracts of epithelial cells. Incubation of the cells and their aqueous cell-free extracts with 125I-labelled parotid saliva resulted in the formation of an iodinated high-molecular-mass complex which was not present in 125I-labelled saline alone. Formation of this complex was time-dependent and was inhibited by treating the buccal epithelial cells or their cell-free extracts with EGTA, iodoacetamide, N-ethylmaleimide or by heating at 100 degrees C for 15 min. The epithelial cells also promoted incorporation of [14C]putrescine into high-molecular-mass complexes whose formation was inhibited by iodoacetamide, unlabelled putrescine and EGTA. Cell extracts mediated cross-linking of monodansylcadaverine into alpha-casein, and this interaction was inhibited by iodoacetamide. Significant amounts of radioactivity were recovered with the epithelial-cell envelopes after exhaustive extraction of 125I-saliva- or [14C]putrescine-treated epithelial cells with 4% (w/v) SDS/10% (v/v) beta-mercaptoethanol. The incorporation of radioactivity into epithelial-cell envelopes was inhibited by pretreatment of the cells with putrescine, EGTA, iodoacetamide, or heating at 100 degrees C for 15 min. These data suggest that: (1) oral mucosal pellicle is formed by the selective adsorption of saliva to the epithelial-cell plasma membrane and its associated cytoskeleton; and (2) the adsorbed salivary components may be cross-linked to each other or the epithelial cytoskeleton by epithelial transglutaminases.     

The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

“On A Piece of Chalk”-Updated1

ABSTRACT. Many advances have been made in our knowledge of the biology of foraminifera over the past several decades. Fine structural, biophysical, and molecular biological studies have shown that the most prominent components of their distinctive bidirectional granuloreliculopods are bundles of micro tubules linked by crossbridges to each other, as well as to membrane-bound organelles and the plasma membrane. the microtubules ratchet past each other as dynein transduces the free energy of ATP to produce pseudopodal movements. In spite of the fact that there are over 40,000 described species of living and fossil species of foraminifera, there have been many recent exciting discoveries of new species and groups. New casting techniques are providing us with greater understanding of the complexities and functional aspects of form in the group. Significant advances are being made in understanding the distribution and energetics of deep-sea forms. Larger and planktonic foraminifera are the hosts for a particularly diverse range of endosymbiotic algae, including dinoflagellates, chlorophytes, unicellular rhodophytes, and diatoms. Chloroplast husbandry also occurs. Significant research effort has been expended yielding us considerable insight into various aspects of the endosymbiotic phenomenon. A unified conceptual framework has been drawn to help us understand the life cycle options found in foraminifera.     

A stem-loop "kissing" model for the initiation of recombination and the origin of introns

Mutations which improve the efficiency of recombination should affect either the proteins which mediate recombination or their substrate, DNA itself. The former mutations would be localized to a few sites. The latter would be dispersed. Studies of hybridization between RNA molecules have suggested that recombination may be initiated by a homology search involving the "kissing" of the tips of stem loops. This predicts that, in the absence of other constraints, mutations which assist the formation of stem loops would be favored. From comparisons of the folding of normal and shuffled DNA sequences, I present evidence for an evolutionary selection pressure to distribute stem loops generally throughout genomes. I propose that this early pressure came into conflict with later local pressures to impose information concerning specific function. The conflict was accommodated by permitting sections of DNA concerned with a specific function to evolve in dispersed segments. Traces of the conflict seem to be present in some modern intron-containing genes. Thus, introns may have allowed the interspersing of selectively advantageous stem loops in coding regions of DNA.      

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.