Assessment of cytochrome P450 sequences offers a useful tool for determining genetic diversity in higher plant species.

To investigate and develop new genetic tools for assessing genome-wide diversity in higher plant-species, polymorphisms of gene analogues of mammalian cytochrome P450 mono-oxygenases were studied. Data mining on Arabidopsis thaliana indicated that a small number of primer-sets derived from P450 genes could provide universal tools for the assessment of genome-wide genetic diversity in diverse plant species that do not have relevant genetic markers, or for which, there is no prior inheritance knowledge of inheritance traits. Results from PCR amplification of 51 plant species from 28 taxonomic families using P450 gene-primer sets suggested that there were at least several mammalian P450 gene mammalian-analogues in plants. Intra- and inter- specific variations were demonstrated following PCR amplifications of P450 analogue fragments, and this suggested that these would be effective genetic markers for the assessment of genetic diversity in plants. In addition, BLAST search analysis revealed that these amplified fragments possessed homologies to other genes and proteins in different plant varieties. We conclude that the sequence diversity of P450 gene-analogues in different plant species reflects the diversity of functional regions in the plant genome and is therefore an effective tool in functional genomic studies of plants.

Quantitative assessment of DNA microarrays--comparison with Northern blot analyses.

DNA microarray is a powerful technology that provides the expression profile of thousands of genes. However, less attention has been paid to its quantitative aspect. In this study, we constructed a small-scale DNA microarray that contains 84 genes and characterized its quantitative aspect. Analyses with this microarray showed that 17 genes were induced, whereas 8 genes were suppressed at least twofold during the differentiation of mouse embryonic stem cells. When repeated with the same combination of fluorescent dyes for probe labeling, the microarray produced consistent data (correlation coefficient = 0.991). In contrast, data were less consistent when repeated with the reverse combination of dyes (correlation coefficient = 0.945). The effect of dye combination was particularly evident in several genes. Total RNA (15 microg) and poly(A) RNA (0.5 microg) showed comparable sensitivity and produced essentially identical data (correlation coefficient = 0.983). The sensitivity of the DNA microarrays was slightly inferior to that of Northern blot analyses. In most genes, data obtained with the two methods were consistent. However, in 4 of 46 genes compared, DNA microarrays failed to detect the expression changes that were revealed by Northern blot. These data demonstrated that DNA microarrays provide quantitative data comparable to Northern blot in general, but a few issues must be considered when analyzing data.

A simple method for screening assessment of skin and eye irritation.

A stepwise, simple screening test for skin and eye irritations, suitable for industrial chemicals or pesticides which are not required to be examined for their exact potential irritancy levels, was developed. The efficacy of the test was evaluated using 15 chemicals including typical irritants (acetic acid, ammonia, chloroacetic acid, dioxane, ethanolamine, formaldehyde, formic acid, hydrogen peroxide, phenol, phosphoric acid, propionic acid, sodium hydroxide, sodium hypochlorite, sulfuric acid, and trichloroacetic acid). Chemicals were chosen so as to represent irritants which act by different mechanisms (i.e., strongly acidic, alkaline, reactive to protein, oxidizing etc.). The method consisted of physicochemical tests and animal tests using rats, mice or guinea pigs, namely, a skin irritation test, an intradermal reaction test and an eye irritation test in a sequential manner such that further tests are not required if a positive result is obtained in earlier steps. Results obtained between two laboratories using this method were very similar. Comparison of our results with the data obtained by the conventional method using rabbits showed fairly good coincidence. The method was shown to be useful in assessing skin and eye irritation of chemicals and causes minimal suffering to animals.

A simple method for screening assessment of acute toxicity of chemicals.

We proposed a simple method for screening assessment of acute oral and dermal toxicity using only three rats and mice of each sex at each dose level. Animals were first treated with chemicals at a dose of 2000 mg/kg and were carefully observed for compound-related morbidity and mortality. If none of the animals died, the following toxicity tests were suspended. If some of the animals died, toxicity tests at doses of 200 and 20 mg/kg were performed. The approximate LD50 values calculated by this method showed little difference between two separate laboratories and were in good agreement with LD50 values reported in the literature. Our toxicological data also showed that LD50 values were about 2-2.5 times the MNLD (maximum non lethal dose) in acute oral and dermal toxicity. This meant that a chemical could be regarded as having an LD50 of about 4000 mg/kg or higher when there was no mortality at the dose of 2000 mg/kg. A chemical with such low toxicity would not require further testing for lethal effects. Therefore, this simple method combining the fixed-dose procedure with the limit test is suitable for determination of approximate LD50 values of chemicals and for screening for necessity for classical full LD50 test using many animals.

[Assessment of the viability of preserved hearts--birefringence test using a polarizing microscope].

The viability of preserved hearts was assessed using the quantitative polarizing microscopic technique or the birefringence test introduced by Braimbridge et al. To clarify the correlation between myocardial degeneration and the change of birefringence, twelve rat hearts under various preserved conditions were examined. To evaluate usefulness of the birefringence test in heart transplantation, thirteen pairs of mongrel dogs were subjected to heterotopic heart transplantation. Among these thirteen, seven were transplanted immediately and another six were transplanted following preservation in cold Collins-M solution for 12 hours. Biopsy specimens were taken at the time of harvest, after preservation, immediately after and 2 hours after transplantation and examined by a polarizing microscope which could measure the change in birefringence following addition of ATP. They were also examined electron-microscopically for degenerative changes. The results of the birefringence test correlated well with the change of the electron microscopic observation and the hemodynamic activities of the transplanted hearts. Thus, it was concluded that the birefringence test was a useful method to evaluate myocardial viability for transplantation within a short time.