Ratio of naturally retained 15N to 13C in rat brain regions as a marker of brain function and activity.

Our aim in the present study was to clarify the activity-dependent and function-associated retention of stable isotopes (SIs) in rat brain regions. We measured regional distributions of the natural stable isotopes 15N and 13C in brain using a mass spectrometer with a dual inlet system and a double collector for ratiometry, and compared them with distributions obtained from internal organs and skeletal muscle. Although levels of 15N and 13C were very high in brain regions of prenatal rats, and robustly decreased after birth, developmental changes in brain regions became obvious when the ratio of 15N to 13C (abbreviated as 15N/13C) in each brain region was compared. A high correlation was observed between free motor activity and 15N/13C in the hippocampus, cerebrum, and striatum. A significantly higher 15N/13C was also observed in the hippocampus and striatum of rats with higher intelligence, which was evaluated by radial maze learning. Furthermore, 15N/13C in brain regions of trained rats were significantly higher than those of untrained age-matched rats. Our study suggests that the 15N/13C in a specific brain region may reflect the physiological feature of the region. This ratio may hence be applicable as a maker for pathological research on undiagnosed brain diseases.

Carbon isotopic signature reveals the geographical trend in methane consumption and production pathways in alpine ecosystems over the Qinghai-Tibetan Plateau.

On the Qinghai-Tibetan Plateau, isotopic signatures in soil-atmosphere CH4 fluxes were investigated in nine grasslands and three wetlands. In the grasslands, the fractionation factor for soil CH4 uptake, αsoil, was much smaller than the usually reported value of 0.9975-1.0095. Stepwise multiple variation analysis indicates that αsoil is higher for higher soil water contents but is lower for higher C/N ratios of soil surface biomass. In the three wetlands, the soil-emitted δ13C-CH4 was similar (-55.3 ± 5.5 ‰ and -53.0 ± 5.5 ‰) in two bogs separated by >1000 km but was lower (-63.4 ± 6.3 ‰) in a marsh. Environmental factors related to intrasite variations in soil-emitted δ13C-CH4 include the soil C/N ratio, oxidation-reduction potential, soil C concentration and soil water contents. Geographical isotopic surveys revealed environmental constraints on the CH4 consumption pathways in grasslands and the biome type-specific consistency in CH4 production pathways in wetlands.

Directional Transport of a Bead Bound to Lamellipodial Surface Is Driven by Actin Polymerization.

The force driving the retrograde flow of actin cytoskeleton is important in the cellular activities involving cell movement (e.g., growth cone motility in axon guidance, wound healing, or cancer metastasis). However, relative importance of the forces generated by actin polymerization and myosin II in this process remains elusive. We have investigated the retrograde movement of the poly-d-lysine-coated bead attached with the optical trap to the edge of lamellipodium of Swiss 3T3 fibroblasts. The velocity of the attached bead drastically decreased by submicromolar concentration of cytochalasin D, latrunculin A, or jasplakinolide, indicating the involvement of actin turnover. On the other hand, the velocity decreased only slightly in the presence of 50 µM (-)-blebbistatin and Y-27632. Comparative fluorescence microscopy of the distribution of actin filaments and that of myosin II revealed that the inhibition of actin turnover by cytochalasin D, latrunculin A, or jasplakinolide greatly diminished the actin filament network. On the other hand, inhibition of myosin II activity by (-)-blebbistatin or Y-27632 little affected the actin network but diminished stress fibers. Based on these results, we conclude that the actin polymerization/depolymerization plays the major role in the retrograde movement, while the myosin II activity is involved in the maintenance of the dynamic turnover of actin in lamellipodium.

Isotopomer and isotopologue signatures of N2O produced in alpine ecosystems on the Qinghai-Tibetan Plateau.

RATIONALE: Static-chamber flux measurements have suggested that one of the world's largest grasslands, the Qinghai-Tibetan Plateau (QTP), is a potential source of nitrous oxide (N2O), a major greenhouse gas. However, production and consumption pathways of N2O have not been identified by in situ field measurements. METHODS: Ratios of N2O isotopomers ((14)N(15)N(16)O and (15)N(14)N(16)O) and an isotopologue ((14)N(14)N(18)O) with respect to (14)N(14)N(16)O in the atmosphere, static chambers, and soils were measured by gas chromatography and mass spectrometry in the summer of 2005 and the following winter of 2006 at three typical alpine ecosystems: alpine meadow, alpine shrub, and alpine wetland, on the QTP, China. RESULTS: Site preference (SP) values of soil-emitted N2 O were estimated as 33.7‰ and 30.1‰ for alpine meadow and shrub, respectively, suggesting larger contributions by fungal denitrification, than by bacterial denitrification and nitrifier-denitrification, to N2 O production. Statistical analysis of the relationship between SP and δ(15)N(bulk) values indicated that in alpine meadow, shrub, and wetland sites fungal denitrification contributed 40.7%, 40.0%, and 23.2% to gross N2O production and the produced N2O was reduced by 87.6%, 82.9%, and 92.7%, respectively. CONCLUSIONS: The combined measurements of N2O concentration, flux, and isotopomeric signatures provide a robust estimation of N2O circulation dynamics in alpine ecosystems on the QTP, which would contribute to the development of ecosystem nitrogen cycle model.

A new method for random mutagenesis by error-prone polymerase chain reaction using heavy water.

Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H(2)O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H(2)O with no Mn(2+), (B) 100% H(2)O/0.6mM Mn(2+), (C) 99% D(2)O with no Mn(2+), (D) 99% D(2)O/0.6mM Mn(2+) and (E) 99% H(2)(18)O with no Mn(2+). The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8×10(-3)errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT→GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H(2)O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D(2)O and H(2)(18)O, may be a powerful random mutagenesis technique.

Food webs in Mongolian grasslands: the analysis of 13C and 15N natural abundances.

Overgrazing often lowers species richness and productivity of grassland communities. For Mongolian grassland ecosystems, a lack of detailed information about food-web structures makes it difficult to predict the effects of overgrazing on species diversity and community composition. We analysed the delta13C and delta15N signatures of herbaceous plants, arthropods (grouped by feeding habit), wild and domestic mammals, and humans in central Mongolia to understand the predominant food-web pathways in this grassland ecosystem. The delta13C and delta15N values of mammals showed little variation within species, but varied considerably with slope position for arthropods. The apparent isotopic discrimination between body tissue and hair of mammals was estimated as 2.0 per thousand for delta13C and 2.1 per thousand for delta15N, which was large enough to cause overestimation of the trophic level of mammals if not taken into account when using hair samples to measure isotopic enrichment.

Stable delta(15)N and delta(13)C isotope ratios in aquatic ecosystems.

In the past 20 years, rapid progress in stable isotope (SI) studies has allowed scientists to observe natural ecosystems from entirely new perspectives. This report addresses the fundamental concepts underlying the use of the SI ratio. The unique characteristics of the SI ratio make it an interdisciplinary parameter that acts as a chemical fingerprint of biogenic substances and provides a key to the world of isotopomers. Variations in SI ratios of biogenic substances depend on the isotopic compositions of reactants, the pathways and kinetic modes of reaction dynamics, and the physicochemical conditions. In fact, every biogenic material has its own isotopic composition, its "dynamic SI fingerprint", which is governed by its function and position in the material flow. For example, the relative SI ratio in biota is determined by dietary lifestyle, e.g., the modes of drinking, eating, and excreting, and appears highly regular due to the physicochemical differences of isotopomers. Our primary goal here is to elucidate the general principals of isotope partitioning in major biophilic elements in molecules, biogenic materials, and ecosystems (Wada, E. et al., 1995). To this end, the nitrogen and carbon SI distribution ratios (delta(15)N and delta(13)C, respectively) are used to examine materials cycling, food web structures, and their variability in various kinds of watershed-including aquatic ecosystems to elucidate an "isotopically ordered world".

Isotopic composition of nitrogenous compounds with emphasis on anthropogenic loading in river ecosystems.

Nitrogenous compounds with high delta(15)N values were recently found in human-dominated small rivers in the Lake Biwa area. A detailed survey was performed to determine the distribution and variation of delta(15)N values in nitrogenous compounds in a representative small river (Hebisuna River) that flows into Lake Nishinoko, an inner bay of Lake Biwa. A high delta(15)N value was detected in the lower reaches of the river and the inner bay, most likely due to denitrification. These results strongly suggest that denitrification in small river systems such as the Hebisuna watershed has contributed to (15)N enrichment in the Lake Biwa ecosystem during the past 40 years. We also observed a clear, stepwise, positive correlation between population density and delta(15)N values for particulate organic matter or sediments. These results demonstrate that delta(15)N (POM) and delta(15)N (sediment) are helpful indicators for assessing nitrogen loading from domestic sewage. Moreover, they will aid in the development of new concepts in the environmental capacity of river ecosystems and its relationship to redox conditions. Finally, our data suggest that a population density of 100-200 persons per km(2) is the upper limit for a watershed in which only simple conventional sewage treatment is in effect.

Use of stable nitrogen isotope signatures of riparian macrophytes as an indicator of anthropogenic N inputs to river ecosystems.

Deterioration of aquatic ecosystems resulting from enhanced anthropogenic N loading has become an issue of increasing concern worldwide, and methods are needed to trace sources of N in rivers. Because nitrate from sewage is enriched in 15N relative to nitrate from natural soils, delta(15)N values of stream nitrate (delta(15)Nnitrate) should be an appropriate index of anthropogenic N loading to rivers, as should the delta(15)N values of riparian plants (delta(15)Nplant) because they are consumers of nitrate. We determined the delta(15)N values of stream nitrate and six species of riparian macrophytes in 31 rivers in the Lake Biwa Basin in Japan. We then tested the correlation between these values and various land-use parameters, including the percentage of land used for residential and agricultural purposes as well as for natural areas. These delta(15)N values were significantly positively correlated with land use (%) that had a high N load (i.e., residential or agricultural use) and significantly negatively correlated with forest (%). These findings indicate that delta(15)N values of stream nitrate and riparian plants might be good indicators of anthropogenic inputs of nitrogen.

Estimation of pathways of the production of greenhouse gases in the tropical swamp forest in Thailand by stable isotope investigation.

Dynamics of greenhouse gases (N(2)O and CH(4)) with the dry-wet cycle along with the variation of oxidation-reduction boundaries were investigated in the tropical wetland in monsoon Asia. It was clarified that the production of N(2)O and CH(4) was closely related to the development of a redox boundary in the Bang Nara River systems. An intermittent increase in N(2)O was observed at the beginning of the rainy season, when a large amount of easily decomposable organic matter was introduced into the river. After 10 days, when dissolved oxygen was consumed completely at the middle reaches, the emission of CH(4) became maximal due to the possible occurrence of denitrification. The distribution of stable isotope ratios in N(2)O clearly demonstrated that nitrification is the major process for its production. Furthermore, the production of N(2)O in this study area was found to vary in time and space with changes in the redox boundary along the water flow.

Changes in stable isotopes, lignin-derived phenols, and fossil pigments in sediments of Lake Biwa, Japan: implications for anthropogenic effects over the last 100 years.

We measured stable nitrogen (N) and carbon (C) isotope ratios, lignin-derived phenols, and fossil pigments in sediments of known ages to elucidate the historical changes in the ecosystem status of Lake Biwa, Japan, over the last 100 years. Stable N isotope ratios and algal pigments in the sediments increased rapidly from the early 1960s to the 1980s, and then remained relatively constant, indicating that eutrophication occurred in the early 1960s but ceased in the 1980s. Stable C isotope ratios of the sediment increased from the 1960s, but decreased after the 1980s to the present. This decrease in stable C isotope ratios after the 1980s could not be explained by annual changes in either terrestrial input or algal production. However, when the C isotope ratios were corrected for the Suess effect, the shift to more negative isotopic value in atmospheric CO(2) by fossil fuel burning, the isotopic value showed a trend, which is consistent with the other biomarkers and the monitoring data. The trend was also mirrored by the relative abundance of lignin-derived phenols, a unique organic tracer of material that originated from terrestrial plants, which decreased in the early 1960s and recovered to some degree in the 1980s. We detected no notable difference in the composition of lignin phenols, suggesting that the terrestrial plant composition did not change markedly. However, we found that lignin accumulation rate increased around the 1980s. These results suggest that although eutrophication has stabilized since the 1980s, allochthonous organic matter input has changed in Lake Biwa over the past 25 years.

Effect of agriculture on water quality of Lake Biwa tributaries, Japan.

We investigated the effects of natural environments and human activity on Lake Biwa, central Japan. We determined the concentrations of 19 elements and the compositions of stable S and Sr isotopes in the main tributaries of the lake and compared them with the corresponding values obtained from the lake water during the circulation period. Results of a principal component analysis indicated that the components dissolved in the lower reaches of the tributaries can be divided into group 1 (HCO(3), SO(4), NO(3), Ca, Mg, Sr) and group 2 components (Cl, Br, Na, K, Ba, Rb, Cs). The concentrations of group 1 components were high in the rivers of the southern area, which is urbanized and densely populated, and the eastern area, which consists of plains where agriculture predominates, compared with the rivers of the northern and western areas, which are mostly mountainous and sparsely populated. The concentrations of group 2 components tended to be high in the river water of industrial areas. The delta(34)S values of SO(4) in the river water converged to 0+/-2 per thousand as the SO(4) concentration increased and, excluding the areas where limestone is extensively distributed, as the HCO(3) concentration increased. In particular, both the delta(34)S values (0+/-2 per thousand) and the (87)Sr/(86)Sr ratios (0.7117+/-0.0005) fell within narrow ranges in the small and medium rivers of the eastern plain area, where rice is widely grown. These values agreed respectively with the delta(34)S values of the fertilizers used in the Lake Biwa basin and the soil-exchangeable (87)Sr/(86)Sr in the eastern plain. The characteristics of water quality in the small and medium rivers of the eastern area can be explained by a model in which sulfuric, nitric, and bicarbonic acids generated by the decomposition of agricultural fertilizer and paddy rice selectively leached out alkaline-earth elements adsorbed on the soil and sediments of the plain or dissolved calcium carbonate enriched with Mg and Sr. Compared with tributary waters, the lake water was depleted in NO(3), owing to denitrification, and in Mn, owing to mineralization, which occur under the redox condition of bottom sediments. Excluding NO(3) and Mn, the compositions of both the dissolved elements and the Sr and S isotopes in the water of Lake Biwa can be approximately reproduced by simple mixing of the tributary water, indicating that these components provide effective indices for evaluating the relationship between the waters of the lake and its tributaries.

Dynamics of 15N natural abundance in wood-decomposing fungi and their ecophysiological implications.

Nine species of basidiomycota and one species of ascomycota were grown in an ammonium sulphate media and on beech wood; and the general (15)N dynamic patterns of the hyphae were examined. The fungal body initially became depleted in (15)N in both the types of incubation. However, the underlying mechanisms were quite different, that is, significant fungal (15)N drop on the beech wood is associated with the fungal N reallocation and the uptake of atmospheric ammonia and/or NO(x), in addition to isotope fractionation during assimilation. Although the (15)N values of the wood-decomposing basidiocarps were generally close to the (15)N values of the wood, it does not always indicate that the wood derived N was the sole N source for the fungi throughout the growth periods as shown in our wood-decomposing experiment.

Sulfur and strontium isotope geochemistry of tributary rivers of Lake Biwa: implications for human impact on the decadal change of lake water quality.

To study the deterioration of the water quality in Lake Biwa, Japan, over the last 40 years, we measured the concentrations and isotopic ratios of sulfur and strontium of water in 41 inflowing rivers and one discharging river. The concentrations of SO4 and Sr of inflowing rivers at downstream sites were generally high in the southern urban area and in the eastern area, where a large agricultural plain is situated, but low in the northern and western areas, whose watersheds are mountainous and with low population density. SO4 and Sr concentrations are also lower at upstream sites, which are closer to mountainous areas. Thus, the inflowing river receives large amounts of SO4 and Sr as it flows across the plain, where human activity levels are high. The delta34S or 87Sr/86Sr values of most eastern rivers at downstream sites are lower than those of water in Lake Biwa, and values become more uniform as the proportion of the plain area in the watershed increases. River water in other areas has higher values of delta34S or 87Sr/86Sr than the lake water. This result indicates that the decadal decrease of delta34S and 87Sr/86Sr in the lake water has been caused mainly by the increased flux of SO4 and Sr from rivers in the eastern plain. We assume that in the plain, sulfur, nitrogen, and organic compounds induced by human activities generate sulfuric, nitric, and organic acids in the water, which accelerate the extraction of Sr from bedrocks, leading to the generation of Sr in the river water in the area.

Fatty acid composition of aquatic insect larvae Stictochironomus pictulus (Diptera: Chironomidae): evidence of feeding upon methanotrophic bacteria.

Larvae of the chironomid Stictochironomus pictulus were collected from Lake Biwa, central Japan. Both the fatty acid composition of the total lipid fraction and the carbon stable isotope ratios of whole larvae were determined. Larvae showed delta(13)C values of -57.4 per thousand to -62.4 per thousand, similar to the values of methane recorded from the lake sediments. A high level of monounsaturated fatty acids (MUFAs; approximately 50% of total fatty acids) and an extremely low level of n-3 series polyunsaturated fatty acids (PUFAs) in the total lipids of S. pictulus indicated a predominantly bacterial nutrition for this species. Moreover, chironomid tissues contained large amounts of the Type I methanotroph group-specific fatty acid, 16:1(n-8) (approximately 8% of total fatty acids). This is the first time such a fatty acid biomarker has been described from freshwater invertebrates. The data suggest that S. pictulus larvae directly feed upon methanotrophic bacteria.

Variations in leaf ß13C along a vertical profile of irradiance in a temperate Japanese forest.

The vertical profile of stable carbon isotope ratios (δ13C) of leaves was analyzed for 13 tree species in a cool-temperate deciduous forest in Japan. The vertical distribution of long-term averaged δ13C in atmospheric CO2 (δa) was estimated from δ13C of dry matter from NADP-malic enzyme type C4 plant (Zea mays L. var. saccharata Sturt.) grown at a tower in the forest for 32␣days, assuming constant Δ value (3.3‰) in Z. mays against height. The δa value obtained from δ13C in Z.␣mays was lowest at the forest floor (-9.30 ± 0.03‰), increased with height, and was almost constant above 10␣m (-7.14 ± 0.14‰). Then leaf Δ values for the tree species were calculated from tree leaf δ13 C andδa. Mean leaf Δ values for the three tall deciduous species (Fraxinus mandshurica, Ulmus davidiana, and Alnus hirsuta) were significantly different among three height levels in the forest: 23.1 ± 0.7‰ at the forest floor (understory), 21.4 ± 0.5‰ in lower canopy, and 20.5 ± 0.3‰ in upper canopy. The true difference in tree leaf Δ among the forest height levels might be even greater, because Δ in Z. mays probably increased with shading by up to ∼‰. The difference in tree leaf Δ among the forest height levels would be mainly due to decreasing intercellular CO2 (C i) with the increase in irradiance. Potential assimilation rate for the three tree species probably increased with height, since leaf nitrogen content on an area basis for these species also increased with height. However, the increase in stomatal conductance for these tree species would fail to meet the increase in potential assimilation rate, which might lead to increasing the degree of stomatal limitation in photosynthesis with height.