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Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3’UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3’UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species. more...

A novel 48-membered cyclic lipid, in which two ether and two amide groups serve as a linker between its hydrophilic and hydrophobic moiety, was synthesized starting from D-1,2-O-isopro-pylidene-sn-glycerol. The synthetic scheme is featured by the selective removal of protecting groups, followed by the introduction of mesylate and carboxylic acid, which sequentially leads to the formation of ether and amide bonds, respectively. Transmission electron microscopic observation confirmed that this lipid forms submicrosized helical ribbons. more...

In order to test the impact of Giardia and geohelminthic infection on infant growth faltering in Bangladesh, a randomised double-blind placebo controlled intervention of 36 weeks' duration was conducted in a rural community located 40 km northwest of Dhaka. Infants aged between 3 and 15 months were randomly assigned to either anti-Giardia and anthelminthic treatment, anti-Giardia treatment only, or a control. Weight and supine length were recorded every 4 weeks. Every 12 weeks intestinal permeability (lactulose/mannitol ratio), haemoglobin, plasma albumin, alpha-1-acid glycoprotein, IgG and Giardia-specific IgM (GSIgM) and eggs of the three common geohelminths and G. intestinalis cysts were determined. Data on 222 fully compliant infants were analysed. No significant differences in intestinal permeability, biochemical or anthropometric variables were found between the intervention groups, although there were associations between improvement in small intestinal mucosal function and better weight-for-age and weight-for-height (length) Z-scores. GSIgM titres indicated high endemicity with rapid re-infection of Giardia among infants; over 95% of infants were positive throughout the study, whereas the stool examination showed very few infants with either geohelminth eggs or Giardia cysts. more...

A cyclic glycerophospholipid, in which two glycerols are connected via alkyl chains to form a 64-membered macrocycle, was synthesized. The structural features that differentiate this from our analogous lipids previously reported are two methylene moieties in the alkyl chains, which were incorporated to improve membrane fluidity. Fluorescence recovery after photobleaching revealed that the membranes made from the methylene-containing cyclic lipid demonstrated fluidity at room temperature unlike those of a linear alkyl-chain lipid of equal ring size. more...

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species. more...

Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the origin and migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species. more...

In this paper we describe the microscopic observation of tubules made from cyclic lipids with different alkyl chain lengths (20, 24, and 28 carbon atoms) by using phase-contrast microscopy, transmission electron microscopy, and scanning electron microscopy. Cooling after incubation produced the rod-like lipid self-assemblies. These microscopic observations revealed that there are two types of tubules: closed-end tubules observed in the dispersion of the cyclic lipids containing alkyl chains 20 and 24 carbons long, and open-end tubules observed in the dispersion of the cyclic lipid with alkyl chains 28 carbons long. more...

Tapping-mode atomic force microscopy has been used to investigate the morphology of mixed membranes composed of 1,2-distearoyl-sn-glycero-phosphatidylcholine and an artificial cyclic lipid with 24-carbon alkyl chains. Images of this supported membrane, deposited on mica by the vesicle fusion method, show a heterogeneous structure. The driving forces for the phase separation derived from the cyclic lipid are discussed here. more...

This review introduces surrogate production as a new technique for fish-seed production in aquaculture. Surrogate production in fish is a technique used to obtain the gametes of a certain genotype through the gonad of another genotype. It is achieved by inducing germ-line chimerism between different species during early development. Primordial germ cells (PGCs) are the key material of this technique to induce germ-line chimera. In several species, it has been reported that PGCs differentiated from the blastomeres inherited some maternally supplied mRNA located in the terminal regions of the early cleavage furrows. PGCs from donor species (or strains) are isolated and transplanted into host species to induce the germ-line chimera. Four methods for inducing germ-line chimera are described: blastomere transplantation, blastoderm-graft transplantation, transplantation of PGC from the genital ridge, and transplantation visualised PGC with GFP fluorescence. Several problems preventing the successful induction of germ-line chimera in various fish species are discussed. Surrogate production, however, opens the possibility of efficient fish-seed production and effective breeding and transfer of biodiversity to an aquaculture strain. Conservation and efficient utilisation of genetic resources will be achieved through surrogate production combined with the cryopreservation of PGCs. more...

The present study analyzes the temperature-dependent sex differentiation in goldfish, which have an XX–XY sex determining system. Hatchlings from an XX male–XX female cross were reared at 23 ± 0.5 °C until Day 12 (days after fertilization), and divided into experimental groups reared at 15, 17, 20, 23, 25, 28 or 30 ± 0.5 °C. The proportions of females were 94.6% at 15 °C, 100% at 17 °C, 94.6% at 20 °C, 90.5% at 23 °C, 46.6% at 25 °C, 15.4% at 28 °C, and 7.7% at 30 ± 0.5 °C. This result confirms that genotypic females can be changed to phenotypic males by high temperature treatment. To determine the temperature sensitive period (TSP) of sex differentiation, the sex ratio was measured in groups of larvae shifted reciprocally between 23 and 30 °C as development progressed. The percentage of females increased in test groups shifted from 23 to 30 °C at Days 17, 22 and 27 and decreased in groups shifted from 30 to 23 °C at Day 17 and 22. The end point of the TSP occurs on Day 17 at 30 °C and on Day 22 at 23 °C, implying that the TSP is shortened at higher temperatures. To investigate the effect of fluctuating temperatures on sex differentiation, several one-day temperature pulses from 23 to 30 °C or 30 to 23 °C were applied, during the TSP. The pulse to 23 °C, from the basal temperature of 30 °C, resulted in a decreased percentage of males, however, the pulse to 30 °C, from the basal temperature of 23 °C, had no effect on the sex ratio. These results show that high temperature must be maintained during the TSP in order to produce a high proportion of masculinized genetic female goldfish. In conclusion, water temperature can play an important role in the process of sex differentiation in goldfish, as has been seen in other teleosts and reptiles, and be applied to aquaculture as a tool for control of sex. more...

High sensitivity differential scanning calorimetry is applied to the problem of interaction between phospholipids and cholesterol in bilayers, i.e., whether or not cholesterol shows a preferential interaction with sphingomyelins over glycerophospholipids. The simplest system yet used for exploring this problem is designed–one composed of 1,2-dimyristoyl- sn -glycero-3-phosphatidylcholine (DMPC), chemically and stereochemically pure N -palmitoyl-sphingomyelin ( pure -C16:0-SM), and cholesterol, based on a belief that chemically and stereochemically purity excludes chain length mismatch and chiral interaction that may affect the interaction from this system. Incremental addition of cholesterol to mixtures of DMPC and pure -C16:0-SM with a ratio of 6:4 or 4:6 leads to a continuous shift in endotherm due to DMPC toward a lower temperature. Such results indicate that cholesterol favors pure -C16:0-SM. more...

To clarify to what extent vascular occlusion or shortage of soluble carbohydrate shortens vase life of cut 'Sonia' roses (Rosa hybrida L.), cut flowers were continuously treated with 200 mg·liter-1 8-hydroxyquinoline sulphate (HQS group), 20 g·liter-1 sucrose (Sue group) or 20 g·liter-1 sucrose plus 200 mg·liter-1 HQS (Sue + HQS group), whereas control flowers were kept in water. All cut flowers were kept at 23°C, 70% RH and 12 hr photoperiod, with 10 μmol·m-2·s-1 irradiance. Although all treatments extended the vase life, sucrose was more effective than HQS. Treatment with sucrose promoted unfolding of petals, suppressed the decrease in fresh weight of cut flowers, and inhibited the occurrence of blueing more than did HQS. Water conductivity of stem segments in the control and Sue groups decreased rapidly after harvest, but that in the HQS and Sue + HQS groups was maintained near the initial level for seven days. The number of bacteria in the stem segments increased in the control and Sue groups, but this increase was suppressed by HQS. Glucose, fructose and sucrose concentrations in petals in the Sue and Sue + HQS groups were much higher than those in the control or HQS groups. These results show that decrease in the soluble carbohydrate concentration in petals was more important than vascular occlusion in determining the vase life of cut 'Sonia' roses under our experimental conditions. more...

Cytochrome P450-aromatase (CYP19) is the critical steroidogenic enzyme controlling estrogen biosynthesis. In teleosts, two genes for CYP19 are found with CYP19A1 expressed mainly in the ovary and CYP19A2 expressed in both brain and pituitary. The promoters for these two homologous genes are considerably different. Exposure to estrogenic endocrine disrupting chemicals (EDC) such as ethinylestradiol and nonylphenol resulted in strong up-regulation of the CYP19A2 gene whereas the expression of CYP19A1 was largely unaffected. Moreover, CYP19A2 transcript abundance was shown to vary during the spawning cycle of both the catfish and the zebrafish which implies an association of the CYP19A2 gene in brain-pituitary endocrinology. Together these findings suggest that EDCs could affect the reproductive physiology of fish through disrupting the gene expression of CYP19A2 in the brain. more...

Studies in the Gambia, using the lactulose–mannitol dual-sugar intestinal permeability test (lactulose:mannitol ratio) as a non-invasive way of investigating mucosal damage, have shown that food malabsorption is significantly associated with early growth retardation. In this cross-sectional study, 210 poor urban Nepali children, 0–60 months old, were recruited and measured for height or length and weight, 167 were examined for intestinal permeability and 173 for parasite infection. Weaning and morbidity data were collected from 172 caretakers. Children were mildly stunted (mean height-for-age Z-score −1·45) and underweight (mean weight-for-age Z-score −1·62). The lactulose:mannitol ratio (0·26) was poorer than that of UK children (0·12), but similar to that found in Bengali children of the same age (0·24). Two stages of weaning, the onset supplementary feeding (6 months) and the cessation of breast-feeding (23 months), were shown to have differential impact. In children currently breast-feeding, the duration of supplementation was negatively related to lactose (PPP=0·031), and poorer height-for-age (P=0·024), which was an unexpected result. No significant relationships were found between intestinal permeability and growth, or with morbidity and helminth infection, except in children withGiardia lambliawho had worse lactulose:mannitol ratios than those without (0·43v.0·25 respectively,P=0·014). It is likely that insults to the gut (e.g. Giardia) and challenges to the immune system (weaning) have a different impact in early and late infancy. more...

The effects of pulse treatment with gibberellin A3 (GA3), silver thiosulfate complex (STS), and with or without sucrose on chlorophyll degradation and vase life of cut flowers of Narcissus tazetta var. chinensis were investigated. The 20-hr pulsing treatment with STS extended the vase life of cut flowers of N. tazetta var. chinensis. Pulse treatments with GA3 delayed degradation of chlorophyll, but extended the vase life only slightly, whereas that with sucrose was ineffective, probably because the concentration of soluble carbohydrates in the stems and floral organs other than the petals was already high. Pulse treatments with STS at 0.1mM in combination with GA3 at 0.5 or 1mM were optimum for extending the vase life and preservation of leaf chlorophyll of N. tazetta var. chinensis. more...

Objectives To determine whether antibiotic treatment leads to improvements in growth in prepubertal children in low and middle income countries, to determine the magnitude of improvements in growth, and to identify moderators of this treatment effect. Design Systematic review and meta-analysis. Data sources Medline, Embase, Scopus, the Cochrane central register of controlled trials, and Web of Science. Study selection Randomised controlled trials conducted in low or middle income countries in which an orally administered antibacterial agent was allocated by randomisation or minimisation and growth was measured as an outcome. Participants aged 1 month to 12 years were included. Control was placebo or non-antimicrobial intervention. Results Data were pooled from 10 randomised controlled trials representing 4316 children, across a variety of antibiotics, indications for treatment, treatment regimens, and countries. In random effects models, antibiotic use increased height by 0.04 cm/month (95% confidence interval 0.00 to 0.07) and weight by 23.8 g/month (95% confidence interval 4.3 to 43.3). After adjusting for age, effects on height were larger in younger populations and effects on weight were larger in African studies compared with other regions. Conclusion Antibiotics have a growth promoting effect in prepubertal children in low and middle income countries. This effect was more pronounced for ponderal than for linear growth. The antibiotic growth promoting effect may be mediated by treatment of clinical or subclinical infections or possibly by modulation of the intestinal microbiota. Better definition of the mechanisms underlying this effect will be important to inform optimal and safe approaches to achieving healthy growth in vulnerable populations. more...

A carbohydrate other than sucrose, glucose, fructose and myo-inositol was detected in sepal extracts of Delphinium. This compound was identified as mannitol by 1 H-NMR. Mannitol was the major carbohydrate in all examined organs: the sepal, the other parts of the flower, the stem and leaves. Mannitol as well as glucose (both at 0.55 M), fed to cut Delphinium flowers, similarly delayed the abscission of sepals. 3-O-methyl glucose (3-OMG) and polyethylene glycol 200 at the same molar concentrations had no such effect. The treatment with glucose markedly increased the concentrations of glucose and fructose in the sepals without changing the concentrations of sucrose and mannitol. On the other hand, the treatment with mannitol increased the concentrations of glucose and fructose in addition to mannitol in the sepals, suggesting that mannitol is metabolized in Delphinium flowers. The treatment with 3-OMG increased the concentration of 3-OMG but not other carbohydrates. Mannitol and glucose similarly delayed the increase in ethylene production in flowers, but 3-OMG did not. The sensitivity to ethylene was similarly reduced by the treatment with glucose and mannitol, but not by 3-OMG. These results suggest that the treatment with mannitol, a major carbohydrate in Delphinium, delayed the abscission of sepals by reducing the sensitivity to ethylene. Mannitol further acted, not merely as an osmolyte, but as an apparent source for carbohydrate metabolism in the flower. more...

Factors, accelerating the senescence of cut Eustoma grandiflorum 'Asuka-no-nami' flowers, were investigated in relation to ethylene production. Pollination markedly accelerated petal senescence of cut Eustoma flowers. Petal senescence was also accelerated by crushing the stigma or removing the stigma and style, but not by the removing the pistil. The climacteric increase in ethylene production from flowers, petals, and pistil was significantly accelerated by pollination. Treatment with 2mM silver thiosulphate complex (STS), an ethylene action inhibitor, extended the vase life of pollinated flowers. These results suggest that the acceleration of flower senescence by pollination is mediated by ethylene. more...

Effect of gibberellin A3 (GA3) on the leaf yellowing and vase life of cut flowers of Narcissus tazetta var. chinensis was examined. Chlorophyll contents of the leaves in the cut spikes or cut leaves were measured with a SPAD meter or spectrophotometer. Continuous and pulse treatments (20 hr) of the cut leaves with GA3, especially at 100μM, delayed the degradation of chlorophyll. In cut spikes, the yellowing of leaves was delayed by the treatment with GA3 at 12°C and 23°C, but the effect was greater at 12°C. Both continuous and pulse treatments extended the vase life of the flowers at 23°C, but only continuous treatment did so at 12°C. The treatment with GA3 did not promote the elongation of cut leaves and peduncles. In conclusion, the treatment with GA3 will be useful for improving the vase life of cut N. tazetta var. chinensis flowers. more...

Methyl glucoside andmyo-inositol are present in all organs of rose (Rosa hybridaL.). To investigate the possible role of these carbohydrates in the opening of cut roses, flowers with a 10, 20 or 40-cm-long stem and a single flower bud (about 1.5 cm in diameter) were placed in water and flower opening and changes in sugar content in flowers and stems examined for 7 d. The longer the stem of the cut flower, the larger was the flower diameter. In stems, the concentration of carbohydrates, including methyl glucoside andmyo-inositol markedly decreased before flower opening. In petals, contents of glucose, methyl glucoside andmyo-inositol also decreased before flower opening, but those of fructose, sucrose and xylose did not. When glucose and methyl glucoside were added to the vase water (4%) flower opening was clearly promoted; this was accompanied by an increase in methyl glucoside and fructose concentrations in petals. On the contrary,myo-inositol inhibited flower opening, and this was accompanied by an increase inmyo-inositol and xylose concentrations in petals. These results suggest that methyl glucoside and/or its metabolites are transported into the petal cells, thereby lowering the osmotic water potential and promoting flower opening.Myo-inositol is not readily metabolized, and exogenousmyo-inositol given at a high concentration may act as an extracellular osmolyte, which inhibits water uptake and flower opening. more...

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum. more...

Concentrations of lead, mercury, and cadmium in the liver, kidney, and muscle of four Steller's sea eagles (Haliaeetus pelagicus) and one white-tailed sea eagle (Haliaeetus albicilla) that were found dead or debilitated and subsequently died in Hokkaido, Japan, from 1986 to 1997 were determined. High lead concentrations (>70 μg/g dry weight) were detected in the liver of two Steller's sea eagles and the white-tailed sea eagle. Lead shot was also found in the intestine and gizzard of one Steller's sea eagle and the white-tailed sea eagle, respectively. Mercury and cadmium concentrations in the tissues of these raptors were low, implying that the toxicities of these elements were negligible. These results indicate that eagles in Hokkaido are suffering from secondary poisoning through ingestion of lead shot embedded in the tissue of their prey. more...

Cut roses ( Rosa hybrida L.) cv. Sonia were continuously treated with water (control), 200 mg l −1 8-hydroxyquinoline sulphate (HQS) or 30 g l −1 sucrose plus 200 mg l −1 HQS (Suc+HQS) at 20, 25 and 30°C. Higher temperatures promoted flower opening and shortened vase life. HQS and Suc+HQS both extended the vase life at all temperatures, but Suc+HQS was the more effective treatment. Flower diameter and fresh weight were markedly increased by Suc+HQS, particularly at 20°C. Hydraulic conductance of stem segments from the control treatment decreased rapidly after harvest, but those for HQS and Suc+HQS treatments were maintained near their initial level. Concentrations of glucose, fructose and sucrose in petals were increased by the Suc+HQS treatment, compared with the control or HQS treatment. Correlations between sugar concentrations in petals and maximum flower diameter or vase life were positive. These results show that soluble carbohydrate concentration in petals is an important factor in determining the vase life of cut rose flowers at all temperatures examined and when hydraulic conductance is not reduced. more...

To investigate the role of soluble carbohydrates, particularly methyl glucoside, myo-inositol and xylose, cut 'Sonia' roses were continuously treated with distilled water (control) or a solution containing 30g·liter-1 sucrose and 200mg·liter-1 8-hydroxyquinoline sulfate (HQS). Changes in carbohydrate concentrations in different plants of the cut flowers were determined. This treatment extended the vase life of cut flowers and increased fresh weight of petals. Fructose, glucose, and sucrose were the main carbohydrates in petals, whereas the concentrations of others were lower at harvest. Although xylose concentration was very low at harvest, it increased with time ; the rest of the carbohydrates changed little with time. Fructose, glucose, and sucrose were the main carbohydrates in the remaining flower part, which were slightly affected by the treatment with sucrose plus HQS. Sucrose, fructose and methyl glucoside were main carbohydrates in the stem at harvest ; their concentrations in the control decreased with time but sucrose plus HQS suppressed this decrease. Sucrose was most abundant in leaves, followed by myo-inositol at harvest. The sucrose concentration in the control decreased sharply during first day after harvest but the treatment with sucrose plus HQS suppressed this decrease. On the contrary, the myo-inositol concentration was almost constant with time regardless of the treatment. Treatment with methyl glucoside and xylose markedly promoted flower bud opening, whereas myo-inositol did not. These results suggest that methyl glucoside and xylose are readily metabolized and contribute to flower opening in cut roses. more...

Using data collected in the health promotion project in Saitama Prefecture in 1995, this study examined the relationship between women's bone mineral density and the number of childbirths. The data consisted of bone mineral density measured at Os calcis by ultrasound bone densitometer (Achilles, Luner) and answers to the questionnaire about age, stature, body weight, number of childbirths, and various items concerning lifestyle. Of 1, 173 women studied, 548 experienced menopause and 625 did not. Multiple regression analysis showed the positive correlation between bone mineral density and the number of childbirths among the subjects who experienced menopause and among the whole subjects. Among the primiparous and multiparous women who were over 45 years of age, the more childbirths the higher the mean bone mineral density, though the nulliparous women over 45 years had a higher density. more...

Flower senescence of Torenia fournieri was investigated in relation to ethylene production and sensitivity. Ethylene production of flowers increased with flower senescence ; pistil contributed to climacteric increase of ethylene production whereas other organs such as the petal, stamen, and calyx did not. Flowers were not sensitive to ethylene on the day of anthesis, but developed sensitivity as the flower senesced. Pollination and wounding of stigma markedly accelerated abscission of flowers, which was accompanied by climacteric ethylene production. Climacteric ethylene, following pollination and stigma wounding, was mainly produced by the pistil, particularly the style with stigma. The abscission of the intact, pollinated and stigma-wounded flowers was significantly inhibited by treatment with silver thiosulfate complex (STS), an ethylene action inhibitor. These results clearly show that the flower senescence of Torenia is mainly regulated by ethylene. more...

We demonstrated extension of flower longevity in torenia (Torenia fournieri Lind.) by introducing a fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene in sense or antisense orientation. Torenia was transformed by Agrobacterium-mediated gene transfer. Among primary transformants, 8 sense-gene-introduced plants (average flower longevity 2.7–7.1 days) and 3 antisense-gene-introduced plants (from 2.5–2.7 days) showed significantly greater longevity than did wild-type plants (2.0 days). Analysis of offspring suggested that the introduced gene had been inherited, and that the extended flower longevity was linked to the existence of the gene. Northern blot analysis and measurement of ethylene production demonstrated a reduction in mRNA level and endogenous ethylene production in plants with improved flower longevity. more...

A microtubule-beta-cyclodextrin conjugate was prepared on a kinesin-adsorbed glass surface by chemical and biochemical means. Fluorescence microscope observation and a motility assay indicated that the conjugate simultaneously expressed an inherent motor function and an inclusion property. more...

Skin pigmentation is one of the most variable phenotypic traits in humans. A non-synonymous substitution (rs1426654) in the third exon of SLC24A5 accounts for lighter skin in Europeans but not in East Asians. A previous genome-wide association study carried out in a heterogeneous sample of UK immigrants of South Asian descent suggested that this gene also contributes significantly to skin pigmentation variation among South Asians. In the present study, we have quantitatively assessed skin pigmentation for a largely homogeneous cohort of 1228 individuals from the Southern region of the Indian subcontinent. Our data confirm significant association of rs1426654 SNP with skin pigmentation, explaining about 27% of total phenotypic variation in the cohort studied. Our extensive survey of the polymorphism in 1573 individuals from 54 ethnic populations across the Indian subcontinent reveals wide presence of the derived-A allele, although the frequencies vary substantially among populations. We also show that the geospatial pattern of this allele is complex, but most importantly, reflects strong influence of language, geography and demographic history of the populations. Sequencing 11.74 kb of SLC24A5 in 95 individuals worldwide reveals that the rs1426654-A alleles in South Asian and West Eurasian populations are monophyletic and occur on the background of a common haplotype that is characterized by low genetic diversity. We date the coalescence of the light skin associated allele at 22–28 KYA. Both our sequence and genome-wide genotype data confirm that this gene has been a target for positive selection among Europeans. However, the latter also shows additional evidence of selection in populations of the Middle East, Central Asia, Pakistan and North India but not in South India., Author Summary Human skin color is one of the most visible aspects of human diversity. The genetic basis of pigmentation in Europeans has been understood to some extent, but our knowledge about South Asians has been restricted to a handful of studies. It has been suggested that a single nucleotide difference in SLC24A5 accounts for 25–38% European-African pigmentation differences and correlates with lighter skin. This genetic variant has also been associated with skin color variation among South Asians living in the UK. Here, we report a study based on a homogenous cohort of South India. Our results confirm that SLC24A5 plays a key role in pigmentation diversity of South Asians. Country-wide screening of the variant reveals that the light skin associated allele is widespread in the Indian subcontinent and its complex patterning is shaped by a combination of processes involving selection and demographic history of the populations. By studying the variation of SLC24A5 sequences among a diverse set of individuals, we show that the light skin associated allele in South Asians is identical by descent to that found in Europeans. Our study also provides new insights into positive selection acting on the gene and the evolutionary history of light skin in humans. more...

We assessed the relationship of fecal environmental contamination and environmental enteropathy. We compared markers of environmental enteropathy, parasite burden, and growth in 119 Bangladeshi children (≤ 48 months of age) across rural Bangladesh living in different levels of household environmental cleanliness defined by objective indicators of water quality and sanitary and hand-washing infrastructure. Adjusted for potential confounding characteristics, children from clean households had 0.54 SDs (95% confidence interval [CI] = 0.06, 1.01) higher height-for-age z scores (HAZs), 0.32 SDs (95% CI = -0.72, 0.08) lower lactulose:mannitol (L:M) ratios in urine, and 0.24 SDs (95% CI = -0.63, 0.16) lower immunoglobulin G endotoxin core antibody (IgG EndoCAb) titers than children from contaminated households. After adjusting for age and sex, a 1-unit increase in the ln L:M was associated with a 0.33 SDs decrease in HAZ (95% CI = -0.62, -0.05). These results are consistent with the hypothesis that environmental contamination causes growth faltering mediated through environmental enteropathy. more...

Subclinical environmental enteropathy is associated with malabsorption of fats, carbohydrates, and vitamins A, B12, and folate; however, little information is available on mineral absorption. We therefore investigated the relation between intestinal mucosal function (measured by the lactulose:mannitol permeability test and plasma citrulline concentration), and zinc (Zn) absorption, as estimated by the change in plasma Zn concentration (PZC) following short-term Zn or placebo supplementation.We conducted a randomized, partially masked, placebo-controlled trial among 282 apparently healthy children 6 to 23 months of age in Burkina Faso. After completing baseline intestinal function tests, participants received either 5 mg Zn, as zinc sulfate, or placebo, daily for 21 days.At baseline, mean ± standard deviation PZC was 62.9 ± 11.9 μg/dL; median (interquartile range) urinary lactulose:mannitol (L:M) recovery ratio and plasma citrulline concentrations were 0.04 (0.03-0.07) and 11.4 (9.0-15.6) μmol/L, respectively. Change in PZC was significantly greater in the Zn-supplemented versus placebo group (15.6 ± 13.3 vs 0.02 ± 10.9 μg/dL; P0.0001), and was negatively associated with initial urinary L:M recovery ratio (-1.1 μg/dL per 50% increase in urinary L:M recovery ratio; P = 0.014); this latter relation did not differ between supplementation groups (P = 0.26). Baseline plasma citrulline concentration was not associated with change in PZC.Although altered intestinal permeability may reduce dietary Zn absorption, it likely does not undermine the efficacy of Zn supplementation, given the large increases in PZC following short-term Zn supplementation observed in this study, even among those with increased urinary L:M recovery ratios. more...

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders). more...

To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors. more...

Thyroid-stimulating hormone receptors (TSHRs) are primarily expressed in the thyroid of vertebrates, however recently, transcripts encoding TSHR have been found abundantly in the gonads in a variety of fish species. The purpose of this study is to characterize the channel catfish TSHR and to examine whether the transcript are translated into protein in the gonad or store the transcript as maternal RNA for later use. The cDNA encoding the TSHR was isolated from the channel catfish thyroid but the transcript was determined to be expressed in a number of tissues, including the gonads. In fact, the ovarian expression of TSHR changed significantly during the reproductive season and peaked after the vitellogenic growth phase. Furthermore, the TSHR transcript was also detected in unfertilized eggs but not in fertilized egg of catfish. LM-PAT analysis demonstrated that catfish TSHR transcripts were fully polyadenylated in thyroidal follicles, gonads and unfertilized eggs suggesting that they were translated into protein opposed to being "stored mRNA". Western blot analysis using polyclonal antibodies against the catfish TSHR verified this assumption by visualizing immunoreactive protein in the thyroid, testis, and the post-vitellogenic ovary in abundance. A functional assay clearly showed that the recombinant catfish TSHR was specifically activated by bovine TSH but not by recombinant catfish follicle-stimulating hormone (FSH) and luteinizing hormone (LH). As in other species, the heterologous gonadotropin, hCG, partially activated the receptor. These results suggested that TSHR plays important roles for gametogenesis rather than embryogenesis. more...

An unidentified carbohydrate was isolated from sweet pea (Lathyrus odoratus L. cv. Diana) petals using HPLC. The isolated compound was identified as L-1-O-methyl-myo-inositol, called L-bornesitol, using (1)H-NMR, (13)C-NMR, and CI-MS. L-Bornesitol was distributed in all organs at high concentrations. L-Bornesitol concentration of petals gradually decreased during flower bud development, but the L-bornesitol content increased by about 5 times. more...

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species. more...

This paper describes the main quality control methods for determining human observer measurement error and instrument error focusing on intra-observer and inter-observer technical error of measurements (TEMs) and relative TEM, and the coefficient of reliability (R). R values above 0.95 are indicative of small errors. To compare variances between different variables, the coefficient of variation, a measure of relative variability, is used. The total variation of a character can be partitioned into genetic, environmental, and error components. Determination of the genetic component of variation (heritability) is usually obtained from twin studies or other family studies. A good environment improves the population mean, whereas a poor environment can lower the population mean without any change in the underlying genetic structure. Worked examples of how to calculate TEM, R, and heritability are provided. more...

Use of BMI as a surrogate for body fat percentage is debatable and universal BMI cut-off points do not seem appropriate; lower cut-off points than currently recommended by WHO should be used in some populations, especially in Asia. The adult WHO BMI database indicates that, on average, women are more obese than men, while men are more likely to be pre-obese than women. Urban rates of overweight and obesity are generally higher than rural rates in both sexes. The trend in pre-obesity and obesity over time is generally upward, with very marked increases in the USA and UK in both sexes over the last 10 years. more...

A series of artificial cyclic lipids that mimic archaeal membrane ones has been synthesized. The structural features of these molecules include a longer cyclic framework, in which the alkyl chain length ranges from 24 to 32 in carbon number, which is longer than our first analogous molecule with 20-carbon long alkyl chains [K. Miyawaki, T. Takagi, M. Shibakami, Synlett 8 (2002) 1326]. Microscopic observation reveals that these molecules have a self-assembling ability: hydration of the lipids yields multilamellar vesicles in aqueous solution and monolayer sheets on solid supports. High-sensitivity differential scanning calorimetry (24- and 28-carbon alkyl chain lipids) indicates that (i) the alkyl chain length affects their phase behavior and (ii) the enthalpies of endothermic peaks accompanied by phase transition were considerably lower than those of their monomeric phospholipid analogs. Fluorescence polarization measurements suggest that the membranes made from the 24-carbon alkyl chain lipid have a higher polarization factor than membranes composed of DMPC and DMPC plus cholesterol. These findings imply that the cyclic lipids containing 24- and 28-carbon alkyl chain construct well-organized monolayer membranes and, in particular, that the molecular order of the 24-carbon alkyl chain lipid is higher than that of bilayer membranes in the liquid-ordered phase. more...

Membrane-bound progestin receptors (mPRs) are potential intermediaries in meiotic maturation of fish oocytes and other physiological processes. In this study, gene expression of the mPRs in the ovary of catfish and zebrafish during the reproductive cycle and the hormonal regulation of the expression were investigated. The transcript abundance of catfish mPRalpha gradually increased in conjunction with ovarian growth and then decreased prior to spawning period whereas the ovarian gene expression of mPRbeta varied little throughout the reproductive cycle. In contrast, mPRgamma gene expression peaked early in the mid-vitellogenic stage. The transcript abundance of zebrafish mPRalpha and beta was low in ovarian follicles at early stages of oogenesis and gradually increased after the onset of vitellogenic growth and, thereafter, the gene expression did not vary. Gonadotropic treatment did not modulate the ovarian expression of mPRalpha and beta genes in either catfish or zebrafish. On the other hand, exposure to 17,20beta-dihydroxy-4-pregenen-3-one (the maturation-inducing steroid in this species) resulted in the down-regulation of mPRalpha in catfish ovary whereas gene expression was significantly induced by estradiol-17beta. Taken together, these findings suggest that gonadotropin-induced final oocyte maturation may not require an induction of mPR(s) expression or that the gonadotropin stimulates mPR protein production at the post-transcriptional level, presuming these receptors are indispensable for oocyte maturation. more...

Estradiol plays a key role in the control of many behavioral and physiological aspects of reproduction therefore the expression of cytochrome P450 aromatase (CYP19), the enzyme responsible for the conversion of androgens to estrogens, is of vital interest. The zebrafish, and many other teleosts, have two aromatase genes (CYP19A1 and CYP19A2) that are expressed predominantly in the ovary and brain, respectively, however, the physiological impact of extra-gonadal aromatase has been poorly described. In this study, in situ hybridizations of whole-mount and paraffin sections of adult zebrafish brains, pituitaries, and ovarian follicles showed that CYP19A2 was strongly expressed in the olfactory bulb (OB), ventral telencephalon (TEL), preoptic area (POA), and ventral/caudal hypothalamic zone (HT) of the brain, and in the anterior and posterior lobes of the pituitary. The regional distribution of the CYP19A2 mRNA did not vary with sex however transcript abundance varied within (male “high expressers” had much higher expression in the OB, TEL, and HT than in “low expressers”) and between sexes (higher in OB, TEL, and HT of males than in females). In situ hybridizations of CYP19A1 failed to develop a signal in the brain or pituitary but were detectable by RT-PCR. CYP19A1 was highly expressed in Stage III B follicles (>500 nm) with significantly lower levels in the Stage IV follicles (>680 nm), Stage III A follicles (>350 nm), and Stage I and II follicles (⩽350 μm) which were embedded in connective tissues. The differential expression of the aromatase genes, particularly CYP19A2 in the brain, suggests that the two aromatase genes play different roles in the reproductive behavior and/or physiology of bony fish. more...

A 65-year-old man was admitted to Tokyo Metropolitan Geriatric Medical Center because of dyspnea on effort, loss of appetite and general fatigue. Chest radiography on admission showed niveau formation in, and infiltration around, the bulla. Percutaneous drainage of the intrabullous fluid was performed and Mycobacterium gordonae was repeatedly cultured from it. The patient was treated with RFP, INH and CAM and the intrabullous fluid and infiltration around the bulla was markedly improved. Seven cases of pulmonary infection by M. gordonae have been reported in Japan, but this is the first case of bulla infected by this organism. more...

This paper describes the first preparation of lipid microtubules that are composed of macrocyclic lipids. The lipid contains diacetylenic alkyl chains in a hydrophobic segment and phosphatidylcholine as a hydrophilic headgroup. The structural features of the assembly include: (i) monolayer wall, (ii) micron scale length, (iii) nano scale diameter, and (iv) elasticity. more...

The cDNA encoding the thyroid stimulating hormone (TSH) receptor was isolated from the ventral aorta (thyroid follicles) and in both gonads of the channel catfish. The seasonal changes in ovarian expression of this receptor seem to be correlated to reproduction. TSH, but not LH or FSH, activated the recombinant TSH receptor expressed in COS cells. These results indicate that the isolated cDNA encodes a functional and highly specific TSH receptor. The seasonally regulated expression in the catfish ovary suggests a role for TSH in gametegenesis. more...