Development of a compact ECR ion source for various ion production.

There is a desire that a carbon-ion radiotherapy facility will produce various ion species for fundamental research. Although the present Kei2-type ion sources are dedicated for the carbon-ion production, a future ion source is expected that could provide: (1) carbon-ion production for medical use, (2) various ions with a charge-to-mass ratio of 1/3 for the existing Linac injector, and (3) low cost for modification. A prototype compact electron cyclotron resonance (ECR) ion source, named Kei3, based on the Kei series has been developed to correspond to the Kei2 type and to produce these various ions at the National Institute of Radiological Sciences (NIRS). The Kei3 has an outer diameter of 280 mm and a length of 1120 mm. The magnetic field is formed by the same permanent magnet as Kei2. The movable extraction electrode has been installed in order to optimize the beam extraction with various current densities. The gas-injection side of the vacuum chamber has enough space for an oven system. We measured dependence of microwave frequency, extraction voltage, and puller position. Charge state distributions of helium, carbon, nitrogen, oxygen, and neon were also measured.

Comparison of the expression patterns of two LIM-homeodomain genes, Lhx6 and L3/Lhx8, in the developing palate.

OBJECTIVES: To compare and contrast the gene expression of two LIM-homeobox type transcription factors, Lhx6 and L3/Lhx8, in secondary palate formation. METHODS: In situ hybridization histochemistry with digoxygenin (DIG) labelled cRNA probes specific for Lhx6 and L3/Lhx8. MATERIALS: Serial cryo-sections of embryonic day (E)13.5, 14.5, and 15.5 mice (C57BL/6). OUTCOME MEASURE: Comparison of the signal intensities of NBT/BCIP precipitate by alkaline phosphatase conjugated anti-DIG antibody. RESULTS: From E13.5 to E15.5, Lhx6 and L3/Lhx8 signals are detected in palatal mesenchyme, but the L3/Lhx8 signal is much more intense than the Lhx6 signal. In palatal epithelium, covering the mesenchyme, Lhx6 mRNA is transiently expressed at E14.5, while L3/Lhx8 mRNA expression is never detected throughout the development. CONCLUSION: Lhx6 and L3/Lhx8 functions may be partially redundant in the mesenchyme of the secondary palate, but not in the palatal epithelium.

Expression of the novel transcription factor OASIS, which belongs to the CREB/ATF family, in mouse embryo with special reference to bone development.

The OASIS gene, which encodes a novel CREB/ATF family member, was isolated from long-term cultured astrocytes that were employed as an in vitro gliosis model. In the present study, we examined the expression pattern of the OASIS gene in the developing mouse embryo by in situ hybridization histochemistry and compared it with the expression of osteogenesis markers. OASIS mRNA expression was most strongly detected in preosteoblasts of the outer bony cortex of the ribs. Alveolar bone also showed strong signals for OASIS gene expression. OASIS mRNA was also localized to the preodontoblast of tooth buds. Expression began at embryonic day 12 (D12.5), peaked around D14.5-16.5, and continued to D18.5. The pattern of expression was very similar to that of hXBP-1 mRNA, which encodes another CREB/ATF family member. Spatiotemporal patterns of OASIS partly overlapped that of osteopontin, osteonectin, and alpha1 type I procollagen genes. Among these, the time course of OASIS mRNA expression was most similar to that of osteopontin mRNA expression, suggesting that the OASIS protein is involved in the late phase of osteoblast differentiation, as compared to the Cbfa1 that regulates early phases of osteoblast differentiation.

Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients. (2) Chorioallantoic membrane (CAM) test.

A chorioallantoic membrane (CAM) assay evaluates the blood vessel reaction and damage to the CAM of a fertilized hen's egg. Two types of CAM assays, the hen's egg test-chorioallantoic membrane (HET-CAM) method and the chorioallantoic membrane-trypan blue staining (CAM-TB) method, were evaluated as alternative methods to the Draize eye irritation test (Draize test). The validation project was composed of three test phases in which 10, 15 and 14 test chemicals, respectively, were evaluated. The test procedure of the five independent laboratories was controlled under the same standard operating procedure (SOP). The interlaboratory variation was relatively high for both methods. However, the rank correlation was relatively high among the values obtained by the five laboratories. The variation associated with the CAM-TB method was smaller than that of the HET-CAM method, which requires macroscopic observation, suggesting that the objectivity and quantitativeness differs between the assay systems. The average values using these two methods were compared with the maximum average Draize total score (MAS). The correlation coefficient (r) between the HET-CAM scores and the MAS was 0.688. This suggests that a simple linear regression may not be appropriate for HET-CAM. However, the Spearman's rank correlation coefficient (rs) was relatively high (rs=0.802). In contrast, the CAM-TB test results showed a good correlation with the MAS when the test chemicals were classified according to their physical properties (r=0.801, liquid and r=0.926, powder). These results suggest that both the HET-CAM and CAM-TB methods may present alternative method of evaluation of eye irritation despite problems of interlaboratory reproducibility.

COLIPA validation project on in vitro eye irritation tests for cosmetic ingredients and finished products (phase I): the red blood cell test for the estimation of acute eye irritation potentials. Present status.

The red blood cell test (RBC test) is part of the COLIPA Validation Project on Alternatives to Draize Eye Irritation. It shows good intra- and interlaboratory reproducibility (reliability) and represents one of the promising in vitro alternatives of this project with a good fit to prediction models (relevance) for the assessment of acute ocular irritancy caused by certain classes of chemicals (mainly surfactants) and formulations. Results obtained during the period of test development, prevalidation, and validation are summarized. The method is based on that of Pape et al. (1987), Pape and Hoppe (1990) and Lewis et al. (1993). The protocol has two endpoints: cellular lysis and changes in protein conformation which can be correlated with initial events in tissue injury inducing inflammatory responses as assessed by Draize eye irritation scoring. Both endpoints are detected by spectrophotometric changes in the haemoglobin absorption at 541nm. The protocol also includes a set of prediction models (PM). One PM is designed to predict three classes of irritancy (classification model) based on both endpoints and the three other PMs are designed to predict modified maximum average scores (MMAS) by algorithms based on data from cellular lysis only. These three PMs [with prediction intervals (PIs)] are: (i) for surfactant ingredients, (ii) for surfactant containing finished products, and (iii) for both groups together. The three PMs are based on a common algorithm derived from historic data. It is shown that PMs derived from historic data from several laboratories, by the same procedure, also produce a good fit with the presented data. Therefore, participating laboratories concluded that the protocol as used in this formal validation study can be considered to be validated for the estimation of acute eye irritation potential of surfactant-containing finished products.

Further evaluation of the quantitative chorioallantoic membrane test using trypan blue stain to predict the eye irritancy of chemicals.

The feasibility of using the chorioallantoic membrane (CAM) test using fertile hens' eggs as an alternative method to predict eye irritancy was examined for 12 test chemicals, comprising eight liquids, three powders and one emulsion. The judgement of the injurious effects of chemicals was carried out by a trypan blue staining method that was developed to overcome disadvantages arising from the lack of objectivity and quantitativeness in the original CAM test (Luepke's method). The amounts of pigment adsorbed by the CAM showed a good correlation with the scores obtained in the Draize eye irritation test. The results indicate that the trypan blue staining assay using CAM-TBS assay may be useful as an alternative to the in vivo eye irritation test. The next step will be to validate this method on an interlaboratory basis.

An in vitro alternative to the Draize eye-irritation test: Evaluation of the crystal violet staining method.

To evaluate a simple in vitro method as an alternative to the Draize eye-irritation test, 12 surfactants were tested by both of the above methods, and the correlation between the results was examined. The in vitro method used was the crystal violet staining method. HeLa cells or SIRC cells from rabbit cornea were inoculated into each well of 96-well microplates which contained serially diluted test chemicals. They were cultured for 72 hr, and then fixed and stained with crystal violet. After spectrophotometric measurement, the concentrations of test materials that inhibited the absorbance to 50% of the control level (IC(50)) were determined. The in vivo data used for comparison were the maximum corneal score and the maximum total score obtained by the eye test when the substances were tested at a concentration of 10%. The correlation coefficients between the maximum corneal scores of the 12 surfactants and their IC(50)s in HeLa cells and in SIRC cells were -0.821 and -0.816, respectively, while those between the maximum total scores and IC(50)s in both cell lines were -0.855 and -0.863, respectively. All of these values are statistically highly significant, indicating that the crystal violet staining method may have value in predicting the eye irritancy of surfactants. Further interlaboratory validation will, however, be needed.

Quantitative evaluation to predict the eye irritancy of chemicals: Modification of chorioallantoic membrane test by using trypan blue.

The chorioallantoic membrane (CAM) test using fertile hen's eggs has been reported as a promising alternative method to predict the eye irritancy of chemicals. However, the scoring system of the original CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Furthermore, the test requires skill when the effects of test substances are to be graded. To overcome these disadvantages, a more objective means of judging the injurious effects of chemicals was examined as follows: after treatment with the test chemical, the CAM was stained with trypan blue and the amount of pigment adsorbed was measured spectrophotometrically. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the in vivo eye irritation test scores (r = 0.89). The findings suggest that the trypan blue staining method is very simple and reproducible, and thus would be a promising alternative method for predicting the eye irritancy of chemicals. However, further studies will be required to validate this test method with a wide variety of chemicals and formulations.

[Effect of PMU therapy (CDDP, MMC and UFT) against terminal gastric carcinoma. Hokuriku Cisplatin Round-table Conference].

PMU therapy with 75 mg/m2 of CDDP, 10 mg/body of MMC and 400 mg/day of UFT was performed on 57 patients with terminal gastric carcinoma. The treatment resulted in CR in 2, PR in 9, MR in 1, NC in 16 and PD in 9 of 37 patients with evaluable cases, with a response rate of 30% (11/37 patients). In the 11 responders, the mean disease-free interval was 6.6 months with a median interval of 5 months, and the mean survival period was 9.2 months with a median survival period of 6 months, while in the 46 non-responders, the mean survival period was 6.7 months with a median survival period of 5 months. In all patients, the mean survival period was 7.1 months with a median survival period of 6 months. Adverse reactions to the treatment included gastrointestinal disturbance observed in 77%, nephrotoxicity in 14%, and myelotoxicity in 26% of the patients, but all reactions became normalized during the course of observation. These results have led to the conclusion that PMU therapy may be an effective treatment for terminal gastric carcinoma.

Liposomes as an in vitro model for predicting the eye irritancy of chemicals.

Liposomes containing 4-methylumbelliferyl phosphate (Um-P) were prepared using the lipid extracts from bovine eyes and were incubated with seven surface-active agents. The Um-P released from the liposomes by each test agent was hydrolysed with alkaline phosphatase and the resultant 4-methylumbelliferone was assayed spectrofluorometrically. The values for Um-P(50) (the concentration of test material at which 50% of Um-P is released) showed a good inverse correlation with the irritation scores obtained by the Draize eye test. The results suggest that the liposomal assay reported here may be useful as an in vitro model for predicting the eye irritancy of chemicals.

Long-term changes in mitochondrial outer membrane enzymes and microsomal enzymes involved in drug and heme metabolism during 2-acetylaminofluorene feeding.

The changes in mitochondrial and microsomal enzyme activities were examined during the course of 2-acetylaminofluorene (2-AAF) hepatocarcinogenesis. 2-AAF produced the phasic changes, such as the early reduction, back to the control and the re-reduction, in mitochondrial monoamine oxidase (MAO) B and kynurenine 3-hydroxylase activities during the course of its hepatocarcinogenic activity in male Wistar rats. 2-AAF also produced decreases in microsomal cytochrome P-450 content and aminopyrine demethylase activity, while it caused an early significant increase and subsequent decrease in cytochrome b5 content in the liver. The change in microsomal protoheme content totally paralleled that of the hemeproteins. Microsomal heme oxygenase activity was significantly increased during 2-AAF feeding, while mitochondrial delta-aminolevulinic acid synthetase activity tended to decrease under the experimental conditions. These changes in mitochondrial and microsomal enzyme activities were more pronounced in the nodules and hepatomas as compared to those of the surrounding tissues.

The protective effect of cysteine on chemical-induced liver injury in rats.

The possible protective effect of cysteine on chemical-induced liver injury was studied in rats in vivo and in vitro. There was no increase in the activity of serum glutamic oxaloacetic transaminase (GOT) of rats pretreated with cysteine (1.2 g/kg, p,o.) followed by 0.25 ml/kg carbon tetrachloride (CCl4), d-galactosamine (GalN) or alpha-naphthylisothiocyanate (ANIT). However, rats pretreated with cysteine followed by 0.5 ml/kg CCl4 were not protected. The content of cytochrome P-450, activity of aminopyrine N-demethylase or serum ratio of 5,5-dimethyl-2,4-oxazolidinedione (DMO) to trimethadione (TMO) (DMO/TMO ratio) after CCl4, GalN or ANIT were not altered by pretreatment with cysteine. However, pretreatment with cysteine prevented changes in the content of cytochrome P-450, activity of aminopyrine N-demethylase and DMO/TMO ratio in serum as well as the activities of serum GOT and GPT when the rats were treated with bromobenzene (BZ). The degree of lipid peroxidation from CCl4 was markedly reduced by the presence of 10(-4)M cysteine. These results suggest that cysteine has a protective effect on chemical-induced liver injury produced via epoxide metabolites.

[The pathogenesis of diabetes produced by subtotal pancreatectomy, with special reference to the cell kinetics of the islet cells].

The pathogenesis of the diabetes produced by the subtotal pancreatectomy was investigated from the pancreatic endocrine function and the cell kinetics of the islet cells. The authors prepared the following two groups in the rats. The first group was 90% pancreatectomized rats and the second group was the 60% pancreatectomized rats. Most of the 90% pancreatectomized rats developed diabetes twelve weeks after operation. In these diabetic rats the insulin secretion was undetectable during glucose infusion. To the contrary, the relative increase of the glucagon secretion was found during arginine infusion in spite of the hyperglycemia. However, non of the 60% pancreatectomized rats developed diabetes during post-operative course up to 6 months. To investigate the cell kinetics of the islet cells, the 3H-thymidine autoradiography was prepared at several intervals after operation. The results of the thymidine labelling index showed two phase of regeneration. The first phase was observed on three to seven days after pancreatectomy and the second phase was on fourteen to twenty-eight days. After the second phase of regeneration, the degeneration of the B-cells was observed in the 90% pancreatectomized rats. On the ninetieth days after operation the atrophy of the islets and the disturbance of the arrangement of A, B and D cells were observed by the immuno-histochemical studies. Meanwhile, the 60% pancreatectomized rats did not show degeneration of the islets. These results show that the onset of diabetes after subtotal pancreatectomy was produced by degeneration of the B cells surpassing their regenerative capacity.