Approximate Bayesian computation analysis of EST-associated microsatellites indicates that the broadleaved evergreen tree Castanopsis sieboldii survived the Last Glacial Maximum in multiple refugia in Japan.

Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.

Investigation of SO3 absorption line for in situ gas detection inside combustion plants using a 4-µm-band laser source.

We have investigated 4-µm-band SO3 absorption lines for in situSO3 detection using a mid-infrared laser source based on difference frequency generation in a quasi-phase-matched LiNbO3 waveguide. In the wavelength range of 4.09400-4.10600 µm, there were strong SO3 absorption lines. The maximum absorption coefficient at a concentration of 170 ppmv was estimated to be about 3.2×10-5 cm-1 at a gas temperature of 190°C. In coexistence with H2O, the reduction of the SO3 absorption peak height was observed, which was caused by sulfuric acid formation. We discuss a method of using an SO3 equilibrium curve to derive the total SO3 molecule concentration.

The population demography of Betula maximowicziana, a cool-temperate tree species in Japan, in relation to the last glacial period: its admixture-like genetic structure is the result of simple population splitting not admixing.

Conservation of the local genetic variation and evolutionary integrity of economically and ecologically important trees is a key aspect of studies involving forest genetics, and a population demographic history of the target species provides valuable information for this purpose. Here, the genetic structure of 48 populations of Betula maximowicziana was assessed using 12 expressed sequence tag-simple sequence repeat (EST-SSR) markers. Genetic diversity was lower in northern populations than southern ones and structure analysis revealed three groups: northern and southern clusters and an admixed group. Eleven more genomic-SSR loci were added and the demographic history of these three groups was inferred by approximate Bayesian computation (ABC). The ABC revealed that a simple split scenario was much more likely than isolation with admixture, suggesting that the admixture-like structure detected in this species was due to ancestral polymorphisms. The ABC analysis suggested that the population growth and divergence of the three groups occurred 96 800 (95% CI, 20 500-599 000) and 28 300 (95% CI, 8700-98 400) years ago, respectively. We need to be aware of several sources of uncertainty in the inference such as assumptions about the generation time, overlapping of generations, confidence intervals of the estimated parameters and the assumed model in the ABC. However, the results of the ABC together with the model-based maps of reconstructed past species distribution and palaeoecological data suggested that the modern genetic structure of B. maximowicziana originated prior to the last glacial maximum (LGM) and that some populations survived in the northern range even during the LGM.

Total serum IgE level influences oral food challenge tests for IgE-mediated food allergies.

Probability curves predicting oral food challenge test (OFC) results based on specific IgE levels are widely used to prevent serious allergic reactions. Although several confounding factors are known to affect probability curves, the main factors that affect OFC outcomes are currently unclear. We hypothesized that an increased total IgE level would reduce allergic reactivity. Medical records of 337 and 266 patients who underwent OFCs for 3.5 g boiled hen's egg white and 3.1 ml raw cow's milk, respectively, were examined retrospectively. We subdivided the patients into three groups based on total IgE levels and age by percentile (<25th, 25-75th, and >75th percentiles), and logistic regression analyses were performed on each group. Patients with higher total IgE levels were significantly less responsive. In addition, age did not significantly affect the OFC results. Therefore, total IgE levels should be taken into account when predicting OFC results based on food-specific IgE levels.

Genome scanning for detecting adaptive genes along environmental gradients in the Japanese conifer, Cryptomeria japonica.

Local adaptation is important in evolutionary processes and speciation. We used multiple tests to identify several candidate genes that may be involved in local adaptation from 1026 loci in 14 natural populations of Cryptomeria japonica, the most economically important forestry tree in Japan. We also studied the relationships between genotypes and environmental variables to obtain information on the selective pressures acting on individual populations. Outlier loci were mapped onto a linkage map, and the positions of loci associated with specific environmental variables are considered. The outlier loci were not randomly distributed on the linkage map; linkage group 11 was identified as a genomic island of divergence. Three loci in this region were also associated with environmental variables such as mean annual temperature, daily maximum temperature, maximum snow depth, and so on. Outlier loci identified with high significance levels will be essential for conservation purposes and for future work on molecular breeding.

Impact of negative frequency-dependent selection on mating pattern and genetic structure: a comparative analysis of the S-locus and nuclear SSR loci in Prunus lannesiana var. speciosa.

Mating processes of local demes and spatial genetic structure of island populations at the self-incompatibility (S-) locus under negative frequency-dependent selection (NFDS) were evaluated in Prunus lannesiana var. speciosa in comparison with nuclear simple sequence repeat (SSR) loci that seemed to be evolutionarily neutral. Our observations of local mating patterns indicated that male-female pair fecundity was influenced by not only self-incompatibility, but also various factors, such as kinship, pollen production and flowering synchrony. In spite of the mating bias caused by these factors, the NFDS effect on changes in allele frequencies from potential mates to mating pollen was detected at the S-locus but not at the SSR loci, although the changes from adult to juvenile cohorts were not apparent at any loci. Genetic differentiation and isolation-by-distance over various spatial scales were smaller at the S-locus than at the SSR loci, as expected under the NFDS. Allele-sharing distributions among the populations also had a unimodal pattern at the S-locus, indicating the NFDS effect except for alleles unique to individual populations probably due to isolation among islands, although this pattern was not exhibited by the SSR loci. Our results suggest that the NFDS at the S-locus has an impact on both the mating patterns and the genetic structure in the P. lannesiana populations studied.

Range shifts of Potentilla fruticosa on the Qinghai-Tibetan Plateau during glacial and interglacial periods revealed by chloroplast DNA sequence variation.

The Qinghai-Tibetan Plateau is one of the most extensive habitats for alpine plants in the world. Therefore, the patterns of genetic variation in populations on the Plateau can reveal the detailed demographic history of alpine plants. We analysed the geographical structure of chloroplast matK sequence variation in Potentilla fruticosa L. (Rosaceae), a shrub currently found across the entire Plateau. We obtained sequence data from 508 individuals from 23 populations at sites ranging from the high-altitude interior to the relatively low-altitude northeastern Plateau. In the interior region, genetic diversity was high and included ancestral haplotypes. In contrast, northeastern populations were characterized by relatively low genetic diversity and recently derived haplotypes. The estimated expansion time in the interior population was 17 times that in the northeastern population. These data suggest that P. fruticosa expanded its range on the Plateau during periods of climatic cooling and contracted to the interior region during warmer periods. Thus, the interior region acted as a refugium and greatly contributed to the diversification of P. fruticosa.

Effects of kin-structured seed dispersal on the genetic structure of the clonal dioecious shrub ilex leucoclada.

Nonrandom patterns of gene dispersal have been identified as possible causes of genetic structuring within populations. Attempts to model these patterns have generally focused solely on the effects of isolation by distance, but the processes involved are more complex than such modeling suggests. Here, we extend considerations of gene dispersal processes beyond simple isolation by distance effects by directly evaluating the effects of kin-structured gene dispersal mediated by the group dispersal of related seeds within fruits (i.e., kin-structured seed dispersal) by birds on genetic structure in Ilex leucoclada, a clonal dioecious shrub. To examine the genetic structure patterns, we established two 30x30 m plots (one with immature soils in old-growth forest and one in secondary forest, designated IM and SC, respectively) with different I. leucoclada stem densities. In these two plots 145 and 510 stems were found, representing 78 and 85 genets, respectively, identified by analyzing their genotypes at eight microsatellite loci. The clonal structure was stronger in the SC plot than in the IM plot. Correlograms of coancestry for genets in both plots exhibited significant, positive, high values in the shortest distance class, indicating the presence of strong genetic structure. However, Sp statistics revealed that the pattern of the genetic structure differed between the plots. In addition, to estimate the family structure within fruits, we sampled forty fruits, in total, from 15 randomly selected plants in the area around the IM and SC plots, and found that 80% of the fruits were multiseeded and 42-100% of the multiseeded fruits contained at least one pair of full sibs. Simulations based on these estimates demonstrated that the group dispersal of related seeds produced through correlated mating both within and across fruits, but not unstructured half-sib dispersal, could generate the observed magnitude and trends of genetic structure found in the IM plot. Furthermore, in addition to kin-structured seed dispersal, isolation by distance processes is also likely to promote genetic substructuring in the SC plot. After discussing possible ecological factors that may have contributed to the observed genetic structure, we contrast our results with those predicted by general isolation by distance models, and propose that kin-structured seed dispersal should promote some evolutionary phenomena, and thus should be incorporated, where appropriate, in models of gene dispersal in natural plant populations.

DNA sequence from a fossil pollen of Abies spp. from Pleistocene peat.

DNA was amplified from individual fossil pollen grains of Abies spp. (Pinaceae), which have been detected from Pleistocene peaty deposits (at least 150,000 years old). To identify the species of the fossil pollen by DNA analysis, the region indicating the species-specific sequence was searched among extant Abies species and the spacer region between rrn5 and trnR in chloroplast DNA was sequenced for four grains of the fossil pollen. Three pollen samples produced the same sequence as extant Abies species. The sequence for the remaining sample differed from that of extant Abies by one substitution. This study showed not only a successful DNA analysis from a single grain of fossil pollen but also a new method to identify the species of fossil pollen for the pollen analysis field.

Liquid chromatographic determination of emamectin, milbemectin, ivermectin and abamectin in crops and confirmation by liquid chromatography-mass spectrometry.

Emamectin, milbemectin, ivermectin and abamectin are similar macrocyclic lactone chemicals used as an acaricides or parasiticides. We developed a simultaneous analytical method for determining the residual amounts of these compounds and emamectin metabolites in crops. A sample extracted with acetone was cleaned up with Bond Elut C18 and NH2. The sample was then fluorescence-derivatized with trifluoroacetic anhydride and 1-methylimidazole in acetonitrile. The analyte was measured by HPLC with fluorescence detection using an octadecylsilyl column with 3 microm particle size and gradient elution. In most crops, their recoveries by the developed method were ca. 80-110%. The detection limits of the analytes in vegetables were 0.1-0.3 ppt. Using the developed method, we surveyed the residues of these compounds in 20 commercial crops in Osaka, Japan. The result of the surveillance was that emamectin benzoate of 0.2-6.7 ppb was detected in nine cases and milbemectin of 16.7-279.3 ppb was detected in four cases. The detected samples were confirmed by LC-electrospray ionization (ESI) MS. The limit of detection by LC-ESI-MS was similar to the fluorescence detection level of 0.1-0.3 ppt in vegetables except for milbemectin.

Degradation of malathion and phenthoate by glutathione reductase in wheat germ.

Residual malathion in wheat was estimated at a lower value when analysis was performed by extraction with acetone after addition of water to swell the wheat, according to the Japanese Bulletin Method. The supernatant of the wheat homogenate showed degradation not only of malathion but also of phenthoate. Malathion and phenthoate were not degraded by the boiled supernatant of the wheat homogenate. It was presumed for this reason that glutathione reductase (GR; EC 1.6. 4.2) in the wheat degraded malathion. The following results were obtained: (1) GR originating in wheat could degrade malathion and phenthoate. (2) The degradation of malathion by the GR was inhibited by excessive GSSG. (3) There was a high correlation between GR activity and malathion degradation activity of the supernatant of wheat homogenates. It is likely that GR acted on the specific structure of malathion and phenthoate, the S=P-S bond, and the blanch structure bonding with the sulfur atom. Following the above, extraction with acetone after addition of water (the Japanese Bulletin Method) should be replaced by extraction with pure organic solvent and without addition of water for swelling.

Determination of pyrethroid residues in vegetables, fruits, grains, beans, and green tea leaves: applications to pyrethroid residue monitoring studies.

Determination of natural pyrethrins and 12 synthetic pyrethroids in agricultural products was investigated. Vegetables and fruits were extracted with acetone, filtered after addition of coagulating solution, partitioned into n-hexane, and cleaned up on a Florisil column (as necessary). Pyrethroids were determined by gas chromatography/electron capture detection (GC/ECD) by using a methyl silicone-coated fused-silica capillary column, and recoveries were calculated by summing peak areas of the components. GC/mass spectrometry was used to identify the pyrethroids detected by GC/ECD monitoring. Grains and beans containing lipids were analyzed by extraction with acetonitrile, partition into n-hexane, Florisil column cleanup, and capillary GC/ECD. The coagulation method was suitable for nonfatty crops such as vegetables, fruits, and green tea leaves, because recoveries were good and Florisil column cleanup was not needed in most cases. The coagulation method was not applicable to lipid-containing crops such as grains and beans because of low recoveries. Recoveries for 18 crops at fortification levels of 0.25-1.0 ppm were 60.0-103.5%. No pyrethroids were detected from the nonfortified crops tested except green tea leaves, in which fluvalinate was detected at 0.89 ppm.

Potential interference by free fatty acids in determination of tricyclazole in brown rice by gas chromatography with flame thermionic detection.

A convenient method is described for the determination of tricyclazole in brown rice, and the interference of free fatty acids with flame thermionic detection (FTD) is reported for the first time. Brown rice is extracted with acetone, the extract is filtered, and the filtrate is evaporated. To the residue is added 10% (w/v) NaCl solution, and the mixture is extracted with ethyl acetate. The extract is charged on a Sep-Pak Plus silica cartridge. Free fatty acids are removed from the rice by washing with diethyl ether, and tricyclazole is eluted with acetone-n-hexane (1 + 1). Tricyclazole is determined on a DB-1 capillary column by gas chromatography with FTD (GC-FTD). Linoleic acid and oleic acid, which have essentially the same retention time as tricyclazole, cannot be detected by FTD. Thus, without the Sep-Pak Plus silica cleanup, the peak height of tricyclazole in the chromatogram decreased, the extent depending on the concentration of linoleic acid. n-Hexane-acetonitrile partitioning was not used for cleanup because it could not remove 50% of the free fatty acids. Recoveries (mean +/- standard deviation, n = 5) of tricyclazole from rice fortified at 2 and 0.1 ppm were 90.5 +/- 9.4% and 81.3 +/- 10.6%, respectively. The limit of quantitation was 0.05 ppm.

Simultaneous determination of residues of emamectin and its metabolites, and milbemectin, ivermectin, and abamectin in crops by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method was developed for the determination of emamectin and its metabolites (8,9-Z-isomer, N-demethylated, N-formylated, and N-methylformylated emamectin) in various crops. The analytes were extracted with acetone, cleaned up on cartridge columns (C18 and NH2), derivatized with trifluoroacetic anhydride and 1-methylimidazole, and determined by LC with fluorescence detection. Because radish inhibited the formation of the fluorescent derivatives, an additional Bond Elut PRS cartridge was used in the cleanup of Japanese radish samples. During sample preparation, N-formylated emamectin partially degraded to emamectin B1b and emamectin B1a, and the 8,9-Z-isomer partially degraded to N-demethylated emamectin. Therefore, emamectin and its metabolites were determined as total emamectin, i.e., their sum was estimated as emamectin benzoate. Their recoveries from most crops were approximately 80-110% with the developed method. The detection limits for the analytes in vegetables were 0.1-0.3 parts per trillion (ppt). The results for these compounds were confirmed by LC/mass spectrometry (LC/MS; electrospray ionization mode). Because the fluorescent derivative of emamectin was undetectable by LC/MS, the results for the analyte were confirmed by using a sample solution without derivatization. Limits of detection by LC/MS were similar to the fluorescence detection limits, 0.1-0.3 ppt in vegetables. In addition to the emamectins, milbemectin, ivermectin, and abamectin were also determined by the developed method.

Determination of clethodim and its oxidation metabolites in crops by liquid chromatography with confirmation by LC/MS.

A method was developed for determination of the herbicide clethodim (C0) and its oxidation metabolites clethodim sulfoxide (C1) and clethodim sulfone (C2) in agricultural products. Upon extraction, both C0 and C1 were oxidized to C2 by m-chloroperoxybenzoic acid, and C2 was determined by liquid chromatography (LC). The C2 peak was confirmed by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI). Recoveries of C0 from radish, tomato, onion, sweet potato, kidney bean, carrot, cabbage, and lettuce ranged from 91 to 118% following fortification at 0.05-1.0 ppm. The detection limit of C2 in crops was 0.01 ppm (S/N > 3). The fortified samples of onion, sweet potato, kidney bean, and carrot were confirmed by LC/MS (ESI), and the peak of C2 was detected.

[Contents of eleven phthalates and di(2-ethylhexyl) adipate in retail packed lunches after prohibition of DEHP-containing PVC gloves for cooking purposes].

Ten samples of retail packed lunches purchased from convenience stores were determined for 11 phthalates and di(2-ethylhexyl) adipate (DEHA) in August 2000, 2 months after the prohibition of DEHP-containing PVC gloves in Japan. Each homogenized sample was extracted with acetonitrile, partitioned with n-hexane, and cleaned up using Florisil and PSA columns. Phthalates in the extract were determined by GC/MS (SIM). The limits of detection were 14.9 ng/g for di(2-ethylhexyl) phthalate (DEHP) and 18.6 ng/g for dibutyl phthalate (DBP). Levels of phthalates in packed lunch samples were 45 to 517 ng DEHP/g (198 ng/g, average), ND to 90 ng DEHA/g, and ND to 10.0 ng BBP/g. Diisononyl phthalate (DINP) was detected in one sample at 76 ng/g. Average DEHP level in ten samples was 4% of that in 1999. The contents of other phthalates were also reduced. DBP was not detected in any sample. Recovery of deuterated isomers added as surrogates was 27.9% for DNP-d4, and 40.6 to 101.5% for the other phthalates.

[Clean-up procedure with ion-exchange mini column in the analysis of flusulfamide in agricultural products by HPLC].

A clean-up procedure with an ion-exchange column in the analysis of flusulfamide by HPLC was examined. Pesticide in the sample was extracted with methanol following liquid-liquid partition with ethyl acetate. The ethyl acetate fraction was cleaned up with silica gel column chromatography. The eluate from the silica gel column was further cleaned up with SAX + PSA mini column, then determined by HPLC. Carotenoids and interfering peaks were removed by washing the combined mini columns with 10 mL of 20% acetone-containing n-hexane and 5 mL of acetone, and flusulfamide was eluted with 35 mL of acetone.

[A case of herniation of the bladder].

A 34-year-old man was admitted with pain on urination, pollakisuria and left inguinal hernia. He had undergone a surgery for the left inguinal hernia 3 times, about 30 years, 28 years and 14 years earlier. Physical examination revealed that there was an elastic soft mass in the left inguinal region. Cystoscopy and cystography showed the bladder herniation and left vesicoureteral reflux (Grade 1). Radical surgery for the hernia of bladder was not performed. The literature on the hernia of the bladder in Japan were collected and discussed.

[A case of emphysematous cystitis].

Emphysematous cystitis is a rare but interesting disease. A case is reported of cystitis emphysema with acute pyelonephritis and uncontrolled diabetes. The patient was a 54-year-old woman complaining of gross hematuria and fever. A submucosa emphysema in bladder was found by cystoscopy . Radiography showed the gas around the bladder. The patient was administered antibiotics, then symptoms was improved. We reviewed 12 cases of emphysematous cystitis reported in Japan and summarized the pathophysiologic features of this entity.

[Clinical study of estramustine phosphate (Estracyt) on prostatic cancer].

Clinical effect of Estracyt was investigated in prostatic cancer patients. Twenty seven patients had been previously treated and 20 had not received prior treatment. Improvement rate of subjective symptoms was 85% in the previously untreated patients and that of objective findings was 85%, while those rates were 44% and 50% in the previously treated patients, respectively. Most of the adverse reactions were changes in mamma and mammary papilla which were considered to be due to the estrogenic activity.