"Live-Autoradiography" Technique Reveals Genetic Variation in the Rate of Fe Uptake by Barley Cultivars.

Iron (Fe) is an essential trace element in plants; however, the available Fe in soil solution does not always satisfy the demand of plants. Genetic diversity in the rate of Fe uptake by plants has not been broadly surveyed among plant species or genotypes, although plants have developed various Fe acquisition mechanisms. The "live-autoradiography" technique with radioactive 59Fe was adopted to directly evaluate the uptake rate of Fe by barley cultivars from a nutrient solution containing a very low concentration of Fe. The uptake rate of Fe measured by live autoradiography was consistent with the accumulation of Fe-containing proteins on the thylakoid membrane. The results revealed that the ability to acquire Fe from the low-Fe solution was not always the sole determinant of tolerance to Fe deficiency among barley genotypes. The live-autoradiography system visualizes the distribution of ß-ray-emitting nuclides and has flexibility in the shape of the field of view. This technique will strongly support phenotyping with regard to the long-distance transport of nutrient elements in the plant body.

Noninvasive imaging of hollow structures and gas movement revealed the gas partial-pressure-gradient-driven long-distance gas movement in the aerenchyma along the leaf blade to submerged organs in rice.

Rice (Oryza sativa) plants have porous or hollow organs consisting of aerenchyma, which is presumed to function as a low-resistance diffusion pathway for air to travel from the foliage above the water to submerged organs. However, gas movement in rice plants has yet to be visualized in real time. In this study involving partially submerged rice plants, the leaves emerging from the water were fed nitrogen-13-labeled nitrogen ([13 N]N2 ) tracer gas, and the gas movement downward along the leaf blade, leaf sheath, and internode over time was monitored. The [13 N]N2 gas arrived at the bottom of the plant within 10 min, which was 20 min earlier than carbon-11 photoassimilates. The [13 N]N2 gas movement was presumably mediated by diffusion along the aerenchyma network from the leaf blade to the root via nodes functioning as junctions, which were detected by X-ray computed tomography. These findings imply the diffusion of gas along the aerenchyma, which does not consume energy, has enabled plants to adapt to aquatic environments. Additionally, there were no major differences in [13 N]N2 gas movement between paddy rice and deepwater rice plants, indicative of a common aeration mechanism in the two varieties, despite the difference in their response to flooding.

Non-invasive 11C-Imaging Revealed the Spatiotemporal Variability in the Translocation of Photosynthates Into Strawberry Fruits in Response to Increasing Daylight Integrals at Leaf Surface.

The efficiency of photosynthate translocation from leaves to fruits directly affects dry matter partitioning. Therefore, controlling photosynthate translocation dynamics is critical for high-yield and high-quality fruit production. Accordingly, photosynthate translocation changes must be characterized using data obtained at a higher spatiotemporal resolution than those provided by conventional methods. In this study, 11C-photosynthate translocation into strawberry (Fragaria × ananassa Duch.) fruits in individual plants was visualized non-invasively and repeatedly using a positron emission tracer imaging system (PETIS) to assess the spatiotemporal variability in the translocation dynamics in response to increasing daylight integrals (i.e., 0.5-, 4.5-, and 9-h exposures to 400 µmol m-2 s-1 at the leaf surface). Serial images of photosynthate translocation into strawberry fruits obtained from the PETIS confirmed that 11C-photosynthates were translocated heterogeneously into each fruit on the same inflorescence. The amount of translocated 11C-photosynthates and the translocation rate into each fruit significantly increased as the integrated light intensity at the leaf surface increased. An analysis of the pedicel of each fruit also confirmed that the photosynthate translocation rate increased. The cumulated photosynthesis in leaves increased almost linearly during the light period, suggesting that an increase in the amount of photosynthates in leaves promotes the translocation of photosynthates from leaves, resulting in an increase in the photosynthate translocation rate in pedicels and enhanced photosynthate accumulation in fruits. Additionally, the distribution pattern of photosynthate translocated to fruits did not change during the light period, nor did the order of the sink activity (11C radioactivity/fruit dry weight), which is the driving force for the prioritization of the 11C-partitioning between competing organs, among fruits. Thus, this is the first study to use 11C-radioisotopes to clarify the spatiotemporal variability in photosynthate translocation from source leaves to individual sink fruits in vivo in response to increasing daylight integrals at a high spatiotemporal resolution.

Alteration of Membrane Fluidity or Phospholipid Composition Perturbs Rotation of MreB Complexes in Escherichia coli.

Gram-negative bacteria such as Escherichia coli are surrounded by inner and outer membranes and peptidoglycan in between, protecting the cells from turgor pressure and maintaining cell shape. The Rod complex, which synthesizes peptidoglycan, is composed of various proteins such as a cytoplasmic protein MreB, a transmembrane protein RodZ, and a transpeptidase PBP2. The Rod complex is a highly motile complex that rotates around the long axis of a cell. Previously, we had reported that anionic phospholipids (aPLs; phosphatidylglycerol and cardiolipin) play a role in the localization of MreB. In this study, we identified that cells lacking aPLs slow down Rod complex movement. We also found that at higher temperatures, the speed of movement increased in cells lacking aPLs, suggesting that membrane fluidity is important for movement. Consistent with this idea, Rod complex motion was reduced, and complex formation was disturbed in the cells depleted of FabA or FabB, which are essential for unsaturated fatty acid synthesis. These cells also showed abnormal morphology. Therefore, membrane fluidity is important for maintaining cell shape through the regulation of Rod complex formation and motility.

Non-invasive imaging of radiocesium dynamics in a living animal using a positron-emitting 127Cs tracer.

Visualizing the dynamics of cesium (Cs) is desirable to understand the impact of radiocesium when accidentally ingested or inhaled by humans. However, visualization of radiocesium in vivo is currently limited to plants. Herein, we describe a method for the production and purification of 127Cs and its use in visualizing Cs dynamics in a living animal. The positron-emitting nuclide 127Cs was produced using the 127I (α, 4n) 127Cs reaction, which was induced by irradiation of sodium iodide with a 4He2+ beam from a cyclotron. We excluded sodium ions by using a material that specifically adsorbs Cs as a purification column and successfully eluted 127Cs by flowing a solution of ammonium sulfate into the column. We injected the purified 127Cs tracer solution into living rats and the dynamics of Cs were visualized using positron emission tomography; the distributional images showed the same tendency as the results of previous studies using disruptive methods. Thus, this method is useful for the non-invasive investigation of radiocesium in a living animal.

Visualising spatio-temporal distributions of assimilated carbon translocation and release in root systems of leguminous plants.

The release of rhizodeposits differs depending on the root position and is closely related to the assimilated carbon (C) supply. Therefore, quantifying the C partitioning over a short period may provide crucial information for clarifying root-soil carbon metabolism. A non-invasive method for visualising the translocation of recently assimilated C into the root system inside the rhizobox was established using 11CO2 labelling and the positron-emitting tracer imaging system. The spatial distribution of recent 11C-photoassimilates translocated and released in the root system and soil were visualised for white lupin (Lupinus albus) and soybean (Glycine max). The inputs of the recently assimilated C in the entire root that were released into the soil were approximately 0.3%-2.9% for white lupin within 90 min and 0.9%-2.3% for soybean within 65 min, with no significant differences between the two plant species; however, the recently assimilated C of lupin was released at high concentrations in specific areas (hotspots), whereas that of soybean was released uniformly in the soil. Our method enabled the quantification of the spatial C allocations in roots and soil, which may help to elucidate the relationship between C metabolism and nutrient cycling at specific locations of the root-soil system in response to environmental conditions over relatively short periods.

On-line rapid purification of [13N]N2 gas for visualization of nitrogen fixation and translocation in nodulated soybean.

Accurate analysis of N fixation in leguminous crops requires determination of N utilization within an intact plant; however, most approaches require tissue disassembly. We developed a simple and rapid technique to generate high-purity and high-yield [13N]N2 gas and obtained real-time images of N fixation in an intact soybean plant. The purification efficiency was ∼81.6% after decay correction. Our method provides accurate signals of N fixation and allows free changes to the tracer gas composition to suit different experimental designs.

Assessment of gamma radiation from a limited area of forest floor using a cumulative personal dosimeter.

To elucidate long term changes in gamma radiation from a limited region of interest of the forest floor, a simple monitoring procedure using a cumulative personal dosimeter (D-shuttle) was examined from 2016 to 2017. The test site was in a small forest in Abiko, Japan, where the initial radiocesium contamination from the Fukushima Dai-ichi Nuclear Power Plant was 60-100 kBq m-2. Three experimental plots basically containing a set of two 5 × 5 m2 observation areas were arranged at the site. The litterfall and decomposing organic layer of one area (D: decontaminated) were fully eliminated before the monitoring, whereas the other area (N: natural) was left unchanged. Five D-shuttle sets (i.e., D-shuttle, lead shield, and holder) per area were set up. One D-shuttle set could monitor the specific gamma radiation from radiocesium distributed within a limited area of ground (0.5 m radius of circle = ca. 0.8 m2 area of flat ground). The results indicated significant differences in the accumulated doses among each of the plots and areas, reflecting their soil radiocesium inventories. Interestingly, every index decreased with time, but the decreases were slower than the theoretical decay of radiocesium (134Cs and 137Cs). In addition, the accumulated dose decreased during heavy rainfall events. One possible explanation for these changes of the accumulated dose is a combination of meteorological and tree phenological phenomena, such as radiocesium from the forest canopy being newly added to the floor primarily by litterfall and soil moisture content disturbing radiation emitted from soils. This simple procedure enables long-term observation of gamma radiation from a limited area of forest floor non-invasively and semi-quantitatively.

Relation between rotation of MreB actin and cell width of Escherichia coli.

Bacterial cells, including Escherichia coli and Bacillus subtilis, continuously elongate and divide. Although the cell width is maintained during cell cycle, the molecular mechanisms involved in its regulation remain unknown. MreB has been implicated to play a role in maintaining cell width. Several point mutations in mreB that affect cell width have been identified. The MreB protein forms clusters or polymers in the cell and moves along annular tracks perpendicular to the long axis. This rotation is coupled with peptidoglycan synthesis. Here, we focused on two MreB mutants, MreBA125V and MreBA174T . Cells producing MreBA125V and MreBA174T were thinner and thicker than WT cells, and MreBA125V and MreBA174T rotated faster and slower than WT MreB, respectively. We observed that the rotation rate correlated with the cell wall synthesis rate. Thus, we conclude that the velocity of MreB rotation also affects cell width, that is, the faster the MreB rotates, the thinner the cell width is.

Dynamic Analysis of Photosynthate Translocation Into Strawberry Fruits Using Non-invasive 11C-Labeling Supported With Conventional Destructive Measurements Using 13C-Labeling.

In protected strawberry (Fragaria × ananassa Duch.) cultivation, environmental control based on the process of photosynthate translocation is essential for optimizing fruit quality and yield, because the process of photosynthate translocation directly affects dry matter partitioning. We visualized photosynthate translocation to strawberry fruits non-invasively with 11CO2 and a positron-emitting tracer imaging system (PETIS). We used PETIS to evaluate real-time dynamics of 11C-labeled photosynthate translocation from a 11CO2-fed leaf, which was immediately below the inflorescence, to individual fruits on an inflorescence in intact plant. Serial photosynthate translocation images and animations obtained by PETIS verified that the 11C-photosynthates from the source leaf reached the sink fruit within 1 h but did not accumulate homogeneously within a fruit. The quantity of photosynthate translocation as represented by 11C radioactivity varied among individual fruits and their positions on the inflorescence. Photosynthate translocation rates to secondary fruit were faster than those to primary or tertiary fruits, even though the translocation pathway from leaf to fruit was the longest for the secondary fruit. Moreover, the secondary fruit was 25% smaller than the primary fruit. Sink activity (11C radioactivity/dry weight [DW]) of the secondary fruit was higher than those of the primary and tertiary fruits. These relative differences in sink activity levels among the three fruit positions were also confirmed by 13C tracer measurement. Photosynthate translocation rates in the pedicels might be dependent on the sink strength of the adjoining fruits. The present study established 11C-photosynthate arrival times to the sink fruits and demonstrated that the translocated material does not uniformly accumulate within a fruit. The actual quantities of translocated photosynthates from a specific leaf differed among individual fruits on the same inflorescence. To the best of our knowledge, this is the first reported observation of real-time translocation to individual fruits in an intact strawberry plant using 11C-radioactive- and 13C-stable-isotope analyses.

First demonstration of multi-color 3-D in vivo imaging using ultra-compact Compton camera.

In the field of nuclear medicine, single photon emission tomography and positron emission tomography are the two most common techniques in molecular imaging, but the available radioactive tracers have been limited either by energy range or difficulties in production and delivery. Thus, the use of a Compton camera, which features gamma-ray imaging of arbitrary energies from a few hundred keV to more than MeV, is eagerly awaited along with potential new tracers which have never been used in current modalities. In this paper, we developed an ultra-compact Compton camera that weighs only 580 g. The camera consists of fine-pixelized Ce-doped Gd3Al2Ga3O12 scintillators coupled with multi-pixel photon counter arrays. We first investigated the 3-D imaging capability of our camera system for a diffuse source of a planar geometry, and then conducted small animal imaging as pre-clinical evaluation. For the first time, we successfully carried out the 3-D color imaging of a live mouse in just 2 h. By using tri-color gamma-ray fusion images, we confirmed that 131I, 85Sr, and 65Zn can be new tracers that concentrate in each target organ.

Synthesis and antimicrobial activity of 2,3-bis(bromomethyl)quinoxaline derivatives.

We synthesized 12 derivatives of 2,3-bis(bromomethyl)quinoxaline with substituents at the 6- and/or 7-positions, and evaluated their activities against bacteria and fungi. Of the 12 compounds, nine (1a-h, 1j, and 1k) showed antibacterial activity. The derivative 1g, which bears a trifluoromethyl group at the 6-position, showed the highest activity against Gram-positive bacteria, while 1c, which has a fluoro-group at the 6-position, showed the widest antifungal activity spectrum. However, only the derivative with an ethyl ester substitution, 1k showed activity against Gram-negative bacteria.

Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having N-Acetyl-d-glucosamine branches.

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear ß-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a ß-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

Branched chitins as nonnatural immunomodulatory biopolymers: secretions of TNF-alpha and NO by direct stimulation of macrophages.

In view of the interesting properties of branched polysaccharides occurring in nature, biological activities of nonnatural branched chitins having beta-1,6-N-acetyl-D-glucosamine branches on the poly(beta-1,4-N-acetyl-D-glucosamine) backbone have been studied. The immunostimulatory activities of the branched chitins were determined and compared with those of lentinan, a beta-1,3-D-glucan having beta-1,6-D-glucose branches, using the mouse macrophagelike cell line RAW264.7 in vitro. The secretions of the tumor necrosis factor and nitric oxide proved to be significantly higher with the branched chitins than with lentinan. Moreover, when interferon-gamma was used in conjunction with the branched chitins on macrophage treatment, a marked augmentation of nitric oxide production was observed. These results are interpreted as the direct stimulation of macrophages by the branched chitins, and the distinctive activities suggest the possibility of developing new types of polysaccharide antitumor agents.

Blood cell separation using amphiphilic copolymers containing N,N-dimethylacrylamide.

To develop leukocyte removal filters effective for whole blood, amphiphilic copolymers based on N,N-dimethylacrylamide were synthesized and evaluated as coating materials for poly(ethylene terephthalate) filters. The copolymers with methyl methacrylate or 2-hydroxypropyl methacrylate as a comonomer showed higher platelet permeation ratios (more than 90%) than that of the copolymer with n-butyl methacrylate, though the logarithmic reductions of leukocytes by these copolymers were less than four. An increase in the platelet permeation for whole blood tended to increase the leukocyte permeation. The permeation of both platelets and leukocytes increased with the amount of copolymer coated on the filters because of the change in the physical properties such as the average pore size, total surface area, and total pore volume of coated filters. These results confirm that both the chemical and physical properties of the filters play important roles to control the permeation behavior.

Chitin and chitosan: functional biopolymers from marine crustaceans.

Chitin and chitosan, typical marine polysaccharides as well as abundant biomass resources, are attracting a great deal of attention because of their distinctive biological and physicochemical characteristics. To fully explore the high potential of these specialty biopolymers, basic and application researches are being made extensively. This review deals with the fundamental aspects of chitin and chitosan such as the preparation of chitin and chitosan, crystallography, extent of N-acetylation, and some properties. Recent progress of their chemistry is then discussed, focusing on elemental modification reactions including acylation, alkylation, Schiff base formation and reductive alkylation, carboxyalkylation, phthaloylation, silylation, tosylation, quaternary salt formation, and sulfation and thiolation.

Preparation and evaluation of trimethylsilylated chitin as a versatile precursor for facile chemical modifications.

Trimethylsilylation of chitin was studied in detail to establish a reliable method, and the properties of the resulting product were elucidated. Chitin was successfully trimethylsilylated with a mixture of hexamethyldisilazane and trimethylsilyl chloride in pyridine. Compared to alpha-chitin, beta-chitin was much more reactive and advantageous as a starting material to prepare fully substituted chitin in a simple manner, though alpha-chitin also underwent full silylation under appropriate conditions. The resulting silylated chitin was characterized by marked solubility in common organic solvents and by easy desilylation to regenerate hydroxy groups, which enabled clean preparation of chitin films. The reactivity of the silylated chitin was examined by treating with triphenylmethyl chloride and acetic anhydride as typical alkylating and acylating reagents, and complete substitutions were readily accomplished. The silylated chitin has thus proved to be a superb precursor for modification reactions.

Nonnatural branched polysaccharides: synthesis and properties of chitin and chitosan having disaccharide maltose branches.

Synthesis and properties of chitin and chitosan derivatives having beta-maltoside branches at C-6 have been studied. Chitosan was first transformed into an organosoluble acceptor having a reactive group only at C-6, 3-O-acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan. Glycosylation with an ortho ester from d-maltose was performed successfully at room temperature in dichloromethane in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst. The degree of substitution could be controlled by the reaction conditions and was up to 0.56. Full deprotection gave chitosan with maltoside branches, and the subsequent N-acetylation resulted in the formation of the corresponding chitin derivative. The introduced disaccharide unit improved hydrophilic properties considerably compared to monosaccharide units as confirmed by high solubility in water and moisture absorption and retention ability. The enzymatic degradability and antimicrobial activity were moderate probably because of the bulky nature of the branches.

Anti-HIV-1 peptides derived from partial amino acid sequences of CC-chemokine RANTES. Regulated upon activation, normal T-cell expressed and secreted.

Fifteen acetyl-peptide-amides with partial amino acid sequences of RANTES (regulated upon activation, normal T-cell expressed and secreted), all Cys residues of which were substituted by Ala, were synthesized, and screened for anti-HIV-1 activity. Peptides corresponding to 1-10, 37-46, and 57-68 showed marked activity against CC-chemokine receptor 5-using HIV-1(JR-CSF) (% inhibition at 100 nM 69, 82, 76%, respectively). The results indicate that multiple regions, including the N-terminal part responsible for chemotactic activity, are involved in anti-HIV-1 activity of RANTES, yielding possible lead compounds for anti-HIV-1 agents.

Synthesis and asymmetric reducing performance of chitin/dihydronicotinamide conjugates having glycine or L-leucine spacer arms.

Chitin/dihydronicotinamide conjugates having glycine or L-leucine spacer arms have been prepared and evaluated as asymmetric reducing agents. N-Nicotinoylglycine and N-nicotinoyl-L-leucine were synthesized and coupled with the amino group of water-soluble 50%-deacetylated chitin. The remaining free amino groups were acetylated, and the nicotinamide groups were transformed into dihydronicotinamide moieties by quaternization followed by reduction. The resulting L-leucine-containing conjugate reduced ethyl benzoylformate efficiently with high chemical yield and asymmetric selectivity, whereas the glycine-containing conjugate gave only poor results. The recovered L-leucine-containing conjugate was reduced to regenerate the dihydronicotinamide structure and could be used again. The L-leucine residue has thus proved suitable as a spacer arm to achieve a high reducing performance.