Prenatal interphase FISH diagnosis of PLP1 duplication associated with Pelizaeus–Merzbacher disease

A submicroscopic genomic duplication in Xq22.2 that contains the entire proteolipid protein 1 gene (PLP1) is responsible for the majority of Pelizaeus–Merzbacher disease (PMD) patients. We previously developed an interphase FISH assay to screen for PLP1 duplications in PMD patients using peripheral blood and lymphoblastoid cell lines. This assay has been utilized as a clinical diagnostic test in our cytogenetics laboratory. To expand usage of the interphase FISH assay to prenatal diagnosis of PLP1 duplications, we examined three PMD families with PLP1 duplications utilizing aminiotic fluid samples. In two families the FISH assay revealed fetuses with PLP1 duplications, whereas the other fetus showed a normal copy number of PLP1. Haplotype analyses, as well as an additional FISH analysis using postnatal blood samples, confirmed the results of the prenatal analyses. Our study demonstrates utility of the interphase FISH assay in the prenatal diagnosis of PLP1 duplications in PMD. Copyright © 2001 John Wiley & Sons, Ltd.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced down-regulation of glucose transporting activities in mouse 3T3-L1 preadipocyte

The possibility that 3T3-L1 preadipocytes, while the level of its glucose uptake activity is relatively low, may offer a useful tool for studying the cause for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced "lipolytic response" was studied. It was established first, that TCDD causes reduction of glucose uptake, one of the hallmark events of the lipolytic process. Then the function of c-Src was investigated. The antisense c-src oligonucleotide decreased the inhibitory action of TCDD on glucose uptake activity in a sequence specific manner. Since antisense oligonucleotides are known to own their blocking effects to their ability to reduce translation of proteins, Western blotting analysis was performed to verify their effectiveness. As expected, the treatment of pre-adipocytes with antisense c-src oligonucleotide reduced c-Src in a sequence specific manner. The treatment of antisense c-src oligonucleotide alone was sufficient to diminish the inhibitory action of TCDD on glucose uptake activity in 3T3-L1 cells, indicating that c-Src is somehow involved in the action of TCDD. In a similar manner, the contribution of c-Fos was investigated using antisense c-fos oligonucleotide, since c-Fos is known to be one of the most affected proteins by c-Src activation among AP-1 members. The treatment of antisense c-fos oligonucleotide did not block the effect of TCDD on glucose uptake activity in 3T3-L1 cells. Therefore, it is unlikely that c-Fos is very important in the lipolytic signal transduction of TCDD mediated through c-Src. In order to determine the relationship between c-Src and c-Myc in the mitotic signal transduction pathway, the effect of antisense c-myc oligonucleotide was investigated. Basically the same result as antisense c-src oligonucleotide experiment was obtained thereby, suggesting the importance of c-Myc as well as c-Src in the signal transduction of TCDD. To show the effect of antisense c-myc oligonucleotide treatment, the level of c-Myc protein by Western blotting and electrophoretic gel-mobility shift assay was assessed. However, antisense c-myc oligonucleotide treatment increased the activity of c-Myc in a sequence specific manner. This may be the result of cellular compensatory response to the initial suppression of c-Myc by antisense treatment. The observation that antisense c-fos oligonucleotide could not block the effect of TCDD indicates that this preadipocyte model is different from the adipocyte differentiation model.     

Hemopoietic cell kinetics after intraperitoneal single injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely spread environmental pollutant. Homopoietic system is one of the targets of TCDD in laboratory animals including monkeys. The present study is the hemopoietic cell kinetics in mice, from the severe depression in cellularity of bone marrow and CFU-GM, to their recovery after the intraperitoneal injection of high dosage of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). The bone-marrow cellularity and CFU-GM were severely decreased to 37.8% and 48% of the control, respectively until day 1 after exposure to TCDD. They were, however, soon recovered, even overshot the control value. Subsequently, they tended to show decrease and oscillation again to and under the control value. In conclusion, our cell kinetic study has proven the oscillation in bone-marrow cellularity and CFU-GM during the recovery period, of which the observation seems to be useful to extend our understanding in the hematotoxicity of TCDD.     

Effect of acid solutions on plants studied by the optical beam deflection method

The optical beam deflection method was applied to study the effects of acid solution on both a terrestial and aquatic plants Egeria and Cerastium, which are common aquatic plant and terrestial weed respectively. A probe beam from a He-Ne laser was passed through a vicinity of a leaf of the plants, which were put in culture dishes filled with acid solutions. Deflection signals of the probe beam were monitored and compared for acid solutions with different pH values. The results of Egria showed that the deflection signals changed dramatically when pH values of acid solutions were 2.0 and 3.0, while little at pH of 4.0 and 5.0. For Cerastium when pH were below 3.0, deflection signals changed greatly with time at the begining. After a certain period of time, deflection signals changed little with time. When pH value was above 4.0, deflection signals of Cerastium were still changing with time even after 20 hours. The results suggested that the damage threshold of pH was between 3.0 and 4.0 for both the land and aquatic plants.     

Seasonal variation of Ra/Ra ratio in seaweed: implications for water circulation patterns in coastal areas of the Noto Peninsula, Japan

To examine water circulation patterns of coastal water, 72 seaweed (Sargasso) samples and 27 coastal water samples were collected from coastal areas of the Noto Peninsula, Japan, during the period from December 1998 to June 2002. The (228)Ra and (226)Ra activities of those samples were measured by low-background gamma-ray spectrometry. There was a wide range of activities of (228)Ra (0.5-2Bq/kg-fresh) and (226)Ra (0.5-1.2Bq/kg-fresh) in the Sargasso samples. The (228)Ra/(226)Ra activity ratio of Sargasso samples exhibited seasonal variation with minimum values in June ((228)Ra/(226)Ra= approximately 1) and maximum values in December (1.5-2.5), which was mainly governed by changes in (228)Ra activity. It is also notable that the seasonal variation of the (228)Ra/(226)Ra ratio of Sargasso is in approximate agreement with that of the ambient coastal water. Sargasso samples appear to have retained the (228)Ra/(226)Ra ratio of the ambient coastal waters, and the temporal variations in that ratio provide insight into seasonal changes in water circulation in the Noto Peninsula coastal area.     

Soil frost control: agricultural adaptation to climate variability in a cold region of Japan

In the northernmost region of Japan (Hokkaido Island), earlier onsets of thick snowcover in recent years (post 1980) have reduced the penetration depth of soil frost, resulting in over-winter survival of unharvested small potato (Solanum tuberosum) tubers that emerge as weeds in the spring in rotation crop fields. To prevent the occurrence of potato weeds, a method was developed to manipulate soil-frost depths by artificially controlling snowcover thickness, guided by a simple numerical model that simulates soil freezing-thawing processes using daily mean air temperature and snowcover thinckness as input variables. The method involves removal of snowcover to expose the soil surface in the beginning of winter until the soil freezes to a sufficient depth. After that time, snow is deposited back or allowed to accumulate naturally to prevent further penetration of frost, which may cause undesirable delay in the seeding of spring crops. Field trials indicated that the model predicted frost depths within several centimeters of observed values, when measured temperature and snowcover thickness were used as model input. Based on the field and laboratory data, a soil temperature of −3°C is necessary for complete elimination of potato tubers. To achieve this temperature in potato-burial zones without causing excessive freezing, an optimal frost depth is 0.3 to 0.4 m. The method is being adopted by progressive potato producers in the region, who use tractor-mounted snow ploughs to manipulate snowcover over a large scale. This is an emerging new technology for agricultural adaptation to climate variability.     

Potential increase in mating frequency of queens in feral colonies of Bombus terrestris introduced into Japan

With the exception of several species, bumblebees are monandrous. We examined mating frequency in feral colonies of the introduced bumblebee Bombus terrestris in Japan. Using microsatellite markers, genotyping of sperm DNA stored in the spermatheca of nine queens detected multiple insemination paternities in one queen; the others were singly mated. The average effective paternity frequency estimated from the genotypes of queens and workers was 1.23; that estimated from the workers’ genotype alone was 2.12. These values were greater than those of laboratory-reared colonies in the native ranges of B. terrestris. The genotypes of one or two workers did not match those of their queens or showed paternities different from those of their nestmates; this may have arisen from either queen takeover or drifting of workers. These alien workers were responsible for the heterogeneous genotype distribution within each B. terrestris colony, resulting in higher estimates of paternity frequency than of insemination frequency. The high mating frequency of introduced B. terrestris may have occurred by artificial selection through mass breeding for commercialization. Moreover, polyandrous queens may be selectively advantageous, because reproduction by such queens is less likely to be disturbed by interspecific mating than that by monandrous queens.     

Current levels of airborne polyfluorinated telomers in Japan

Fluorotelomer alcohols (FTOHs), a class of per- and poly-fluorochemicals (PFCs), are environmental contaminants characterized by high volatility and are therefore prone to long-range atmospheric transport. In this study, airborne FTOHs were identified in Japan using a newly developed passive air sampler containing activated carbon felts. Air sampling was conducted at 33 sites all over Japan from April to June 2007. 8:2 FTOH, the dominant analyte, ranged from <32 to 2466pgm(-3) (mean: 241pgm(-3)) followed by 6:2 FTOH from n.d. to 768pgm(-3) (mean: 51.6pgm(-3)), 10:2 FTOH from <17 to 113pgm(-3) (mean: 26.8pgm(-3)) and 8:2 FTOAcryl from n.d. to 480pgm(-3) (mean: 25.8pgm(-3)). 8:2 FTOMethacryl was not detected above detection limit (3pgm(-3)) in all samples. The highest concentration of total investigated PFCs was found in Chiba (35 degrees 43'20' N 140 degrees 39'46' E) (2152-3181pgm(-3)). On the other hand, in three locations of Higashi-Yodogawa (34 degrees 44' N 135 degrees 32' E), high levels of 8:2 FTOAcryl (239pgm(-3), 109-480pgm(-3)) were detected, the levels of which were comparable to those of 8:2 FTOH (mean: 175pgm(-3), range: 115-292pgm(-3)). This study is the first national wide-scale survey of FTOHs and 8:2 FTOAcryl across Japan, showing that 8:2 FTOH was ubiquitous in the environment in Japan.     

Contribution of metabolites to mutagenicity during anaerobic biodegradation of fenitrothion

The contribution of fenitrothion and its microbial metabolites to the mutagenicity of a fenitrothion-containing solution was investigated during anaerobic biodegradation. Although a mixed culture of bacteria obtained from a paddy field degraded fenitrothion and reduced its concentration from 4.6 to 0.1 mg/l in 6 days, the indirect mutagenicity of the solution in Salmonella strain YG1029 increased. This increase was found to be partially due to amino-fenitrothion generated during the biodegradation. In addition, other unidentified metabolites contributed to the mutagenicity. In contrast, the indirect mutagenicity in strain YG1042, which was initially large because of fenitrothion, then decreased, and increased again. This increase in mutagenicity was also due to amino-fenitrothion and other unidentified metabolites. The mutagenicity in strains YG1029 and YG1042 decreased after day 6. The greatest contribution of amino-fenitrothion to the mutagenicity was calculated to be 73% and 61% in YG1029 and YG1042 on day 3 of incubation, respectively. That of unidentified metabolites was calculated at 49% and 61% on day 20, respectively. Therefore, because not all the toxic metabolites of a compound can be identified, it is important to evaluate the toxicity of a whole solution in a bioassay such as the Ames assay rather than deducing the toxicity of the solution from the combined toxicities of known metabolites.     

Fate of mutagenicity produced during anaerobic biodegradation of the herbicide chlornitrofen (CNP)

The mutagenicity of chlornitrofen (CNP)-containing solutions has been reported to increase during anaerobic biodegradation. In the present study, the fate of this increased mutagenicity under subsequent aerobic and anaerobic incubation conditions was investigated using two Salmonella tester strains, YG 1024 (a frameshift-detecting strain) and YG 1029 (a base-pair-substitution-detecting strain). Mutagenicity for both YG 1024 and YG 1029 strains increased during nine-day anaerobic biodegradation. During subsequent anaerobic incubation, the increased mutagenicity decreased gradually for YG 1029 but did not change significantly for YG 1024. By contrast, the increased mutagenicity decreased rapidly after the conversion to aerobic incubation for both YG 1024 and YG 1029 strains. The rapid decrease in mutagenicity during aerobic incubation was due to decreases, not only in an identified mutagenic metabolite (CNP-amino) but also in unidentified mutagenic metabolites.