Melanin-concentrating hormone receptor 1 is discarded by exosomes after internalization.

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a G protein-coupled receptor, is poised for interaction with its ligands on the plasma membrane. Analyses of MCHR1 knockout mice suggest that this receptor could be a therapeutic target for the treatment of appetite disorders, glucose metabolism, psychiatric disorders, and inflammation. Binding of MCH to MCHR1 initiates calcium signaling, which is subsequently attenuated through receptor internalization. However, the ultimate destiny of the receptor post-internalization remains unexplored. In this study, we report the extracellular secretion of MCHR1 via exosomes. The recruitment of MCHR1 to exosomes occurs subsequent to its internalization, which is induced by stimulation with the ligand MCH. Although a highly glycosylated form of MCHR1, potentially representing a mature form, is selectively recruited to exosomes, the MCHR1 transferred into other cells does not exhibit functionality. The truncation of MCHR1 at the C-terminus not only impairs its response to MCH but also hinders its recruitment to exosomes. These findings imply that functional MCHR1 could be secreted extracellularly via exosomes, a process that may represent a mechanism for the termination of intracellular MCHR1 signaling.

Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276.

Exosomes are small extracellular vesicles (EVs) found in culture supernatants, blood, and breast milk. The size of these nanocomplexes limits the methods of EV analyses. In this study, nitrobenzoxadiazole (NBD), a fluorophore, conjugated endosome-lysosome imager, GIF-2250 and its derivative, GIF-2276, were evaluated for exosome analyses. A correlation was established between GIF-2250 intensity and protein maker levels in bovine milk exosomes. We found that high-temperature sterilization milk may not contain intact exosomes. For precise analysis, we synthesized GIF-2276, which allows for the covalent attachment of NBD to the Lys residue of exosome proteins, and labeled milk exosomes were separated using a gel filtration system. GIF-2276 showed chromatographic peaks of milk exosomes containing >3 ng protein. The area (quantity) and retention time (size) of the exosome peaks were correlated to biological activity (NO synthesis suppression in RAW264.7 murine macrophages). Heat denaturation of purified milk-derived exosomes disrupted these indicators. Proteome analyses revealed GIF-2276-labeled immunomodulators, such as butyrophilin subfamily 1 member A1 and polymeric immunoglobulin receptor. The immunogenicity and quantity of these factors decreased by heat denaturation. When milk exosomes were purified from market-sourced milk we found that raw and low-temperature sterilization milk samples, contained exosomes (none in high-temperature sterilization milk). These results were also supported by transmission electron microscopy analyses. We also found that GIF-2276 could monitor exosome transportation into HEK293 cells. These results suggested that GIF-2250/2276 may be helpful to evaluate milk exosomes.

Simulation of nonlinear propagation of femtosecond laser pulses in air for quantitative prediction of the ablation crater shape.

The utilization of sub-100 fs pulses has attracted attention as an approach to further improve the quality and precision of femtosecond laser microfabrication. However, when using such lasers at pulse energies typical for laser processing, nonlinear propagation effects in air are known to distort the beam's temporal and spatial intensity profile. Due to this distortion, it has been difficult to quantitatively predict the final processed crater shape of materials ablated by such lasers. In this study, we developed a method to quantitatively predict the ablation crater shape, utilizing nonlinear propagation simulations. Investigations revealed that the ablation crater diameters derived by our method were in excellent quantitative agreement with experimental results for several metals over a two-orders-of-magnitude range in the pulse energy. We also found a good quantitative correlation between the simulated central fluence and the ablation depth. Such methods should improve the controllability of laser processing with sub-100 fs pulses and contribute to furthering their practical application to processes over a wide pulse-energy range, including conditions with nonlinear-propagating pulses.

Tissue-free water tritium and non-exchangeable organically bound tritium concentrations in fish near coastline during and after operation of Japan's first nuclear fuel reprocessing facility.

The risks from radioactive wastewater release from nuclear facilities into the ocean are a global concern. Radioactive contaminants, such as tritium (3H), in both forms of tissue free water tritium (TFWT) and non-exchangeable organically bound tritium (NE-OBT), can be incorporated into marine biota and cause radiation doses to biota and future consumers. However, no studies have been conducted to measure both forms of 3H in marine fish as well as evaluate the residence time in the vicinity of a nuclear fuel reprocessing facility. Here, fish from a brackish lake and from the Pacific Ocean coastline of Japan, which are near such a facility, were collected between 2006 and 2021. The reprocessing facility was operational between 2006 and 2009, during which time about 300 times more tritiated water was discharged per year into the ocean compared to the period when the facility was not operational. During operation the annual release was 30 times higher than the treated water released annually from Fukushima Daiichi. As expected, TFWT and NE-OBT concentrations increased in marine fish during operations and had peak values of 3.59 ± 0.03 and 0.56 ± 0.03 Bq/L, respectively. Total dose rates to the fish were 36,000 times lower than the 10 µGy h-1 benchmark. Concentrations gradually decreased to pre-operational levels as the facility was turned off with NE-OBT taking twice as long. Fish sampled from the brackish lake tended to have more incorporated TFWT and NE-OBT concentrations than ocean fish. This indicates that ocean tides might have contributed to the accumulation of discharged tritiated water in the lake via a narrow water channel, which highlights the importance of examining all marine ecosystems in future operations. In both marine environments, the estimated committed effective dose using the highest observed data through ingestion was well below public limits (91,000 times lower).

Temporal variation of tritium concentration in monthly precipitation collected at a Difficult-to-Return Zone in Namie Town, Fukushima Prefecture, Japan.

This article discusses tritium concentrations in monthly precipitation in part of the Difficult-to-Return Zone in Namie Town during 2012-2021. The tritium concentrations, which were measured with a low background liquid scintillation counter after carrying out an enrichment procedure, fluctuated seasonally from 0.10 ± 0.02 to 0.85 ± 0.02 Bq L-1. This range of concentrations is concluded to not be unusual based on comparisons with the concentrations at other sites and estimates of the past range of the concentrations. Moreover, no significant variations in observed tritium concentrations were observed due to decommissioning work at the Fukushima Dai-ichi Nuclear Power Plant. These results contribute to understanding the background level of tritium concentration in precipitation before the oceanic discharge of treated water from the Fukushima plant. In addition, this article evaluates the amount of tritium supplied to the ocean by terrestrial rainwater pouring into the Pacific Ocean via Ukedo River, which flows through Namie Town; this information will contribute to the discussion on the impact of the oceanic discharge of treated water.

Calibration experiments for radon in drinking water measurements using portable-type electrostatic-collection radon monitors.

Portable-type electrostatic-collection radon monitors (RAD7) are often used for in-situ measurements of radon in water. In this study, we evaluated the calibration factors and their uncertainties for two RAD7 monitors based on comparative measurements with the liquid scintillation counting method. In the first experiment, we found that both RAD7 monitors had relatively large uncertainties due to leakage of radon gas that bubbled from the gaps between the lids of the desiccant container and the glass vial. Therefore, for the second experiment, these gaps were closed as much as possible using parafilm and clay, respectively. As a result, the relative uncertainties for both RAD7 monitors were significantly decreased. Furthermore, we collected spring water samples to confirm the reliability of radon concentrations. After closing the leakage point, the uncertainty of radon concentrations in spring water we measured using the typical protocol of the RAD7 were significantly lower, which improves the measurement.

INFLUENCE OF SAMPLING FLOW RATE ON THORON EXHALATION RATE MEASUREMENTS BY THE CIRCULATION METHOD.

Researchers have used various methods to obtain the exhalation rates of radon and thoron from soil and building materials. One of the typical methods for radon exhalation rate is the circulation method using an accumulation container, an external or internal sampling pump and a continuous radon monitor. However, it is necessary to consider sampling flow rate if this method is applied to exhalation rate measurement for thoron due to its short half-life. Based on a calibration experiment, the measured thoron concentrations obtained by an electrostatic collection type radon and thoron monitor (RAD7) were found to be influenced strongly by the sampling flow rate. It was also found that the thoron exhalation rate from a soil sample depended on the pressure difference which was proportional to the increasing sampling flow rate. The thoron exhalation rate measured at the generally used sampling flow rate of the internal sampling pump of the RAD7 was overestimated compared with the value at 0 L min-1.

222Rn and 226Ra Concentrations in Spring Water and Their Dose Assessment Due to Ingestion Intake.

222Rn and 226Ra concentrations of less than a few to several thousands of Bq L-1 have been observed in several underground bodies of water around the world. Although regulations for these concentrations in water have been implemented internationally, there are currently no regulations in place in Japan. However, concentrations that exceed these internationally recognized regulatory values have also been observed in Japan. In this study, concentrations in spring water in the northern part of Japan were measured and the effective dose from intake of the water was evaluated. 222Rn concentrations were measured using a liquid scintillation counter, and 226Ra concentrations were measured using a high purity germanium detector after chemical preparation. The measured 222Rn concentrations (=12.7 ± 6.1 Bq L-1) and 226Ra concentrations (<0.019-0.022 Bq L-1) did not exceed the reference values set by international and European/American organizations. A conservative estimate of the annual effective ingestion dose of 8 µSv for 222Rn and 226Ra obtained in this study is much smaller than the estimated overall annual effective dose of 2.2 mSv from natural radiation to the Japanese population. However, this dosage accounts for 8% of the WHO individual dosing criteria of 0.1 mSv/year for drinking water.

Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats.

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG)( n ) was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/µg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura(+) mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.

Long-Term Measurements of Radon and Thoron Exhalation Rates from the Ground Using the Vertical Distributions of Their Activity Concentrations.

A long-term measurement technique of radon exhalation rate was previously developed using a passive type radon and thoron discriminative monitor and a ventilated type accumulation chamber. In the present study, this technique was applied to evaluate the thoron exhalation rate as well, and long-term measurements of radon and thoron exhalation rates were conducted for four years in Gifu Prefecture. The ventilated type accumulation chamber (0.8 × 0.8 × 1.0 m3) with an open bottom was embedded 15 cm into the ground. The vertical distributions of radon and thoron activity concentrations from the ground were obtained using passive type radon-thoron discriminative monitors (RADUETs). The RADUETs were placed at 1, 3, 10, 30, and 80 cm above the ground inside the accumulation chamber. The measurements were conducted from autumn 2014 to autumn 2018. These long-term results were found to be in good agreement with the values obtained by another methodology. The radon exhalation rates from the ground showed a clearly seasonal variation. Similar to findings of previous studies, radon exhalation rates from summer to autumn were relatively higher than those from winter to spring. In contrast, thoron exhalation rates were not found to show seasonal variation.

Discriminative Measurement of Absorbed Dose Rates in Air from Natural and Artificial Radionuclides in Namie Town, Fukushima Prefecture.

Ten years have elapsed since the accident at the Fukushima Daiichi Nuclear Power Plant in 2011, and the relative contribution of natural radiation is increasing in Fukushima Prefecture due to the reduced dose of artificial radiation. In order to accurately determine the effective dose of exposure to artificial radiation, it is necessary to evaluate the effective dose of natural as well as artificial components. In this study, we measured the gamma-ray pulse-height distribution over the accessible area of Namie Town, Fukushima Prefecture, and evaluated the annual effective dose of external exposure by distinguishing between natural and artificial radionuclides. The estimated median (range) of absorbed dose rates in air from artificial radionuclides as of 1 April 2020, is 133 (67-511) nGy h-1 in the evacuation order cancellation zone, and 1306 (892-2081) nGy h-1 in the difficult-to-return zone. The median annual effective doses of external exposures from natural and artificial radionuclides were found to be 0.19 and 0.40 mSv in the evacuation order cancellation zone, and 0.25 and 3.9 mSv in the difficult-to-return zone. The latest annual effective dose of external exposure discriminated into natural and artificial radionuclides is expected to be utilized for radiation risk communication.

Comprehensive exposure assessments from the viewpoint of health in a unique high natural background radiation area, Mamuju, Indonesia.

Mamuju is one of the regions in Indonesia which retains natural conditions but has relatively high exposure to natural radiation. The goals of the present study were to characterize exposure of the entire Mamuju region as a high natural background radiation area (HNBRA) and to assess the existing exposure as a means for radiation protection of the public and the environment. A cross-sectional study method was used with cluster sampling areas by measuring all parameters that contribute to external and internal radiation exposures. It was determined that Mamuju was a unique HNBRA with the annual effective dose between 17 and 115 mSv, with an average of 32 mSv. The lifetime cumulative dose calculation suggested that Mamuju residents could receive as much as 2.2 Sv on average which is much higher than the average dose of atomic bomb survivors for which risks of cancer and non-cancer diseases are demonstrated. The study results are new scientific data allowing better understanding of health effects related to chronic low-dose-rate radiation exposure and they can be used as the main input in a future epidemiology study.

A unique high natural background radiation area - Dose assessment and perspectives.

The biological effects of low dose-rate radiation exposures on humans remains unknown. In fact, the Japanese nation still struggles with this issue after the Fukushima Dai-ichi Nuclear Power Plant accident. Recently, we have found a unique area in Indonesia where naturally high radiation levels are present, resulting in chronic low dose-rate radiation exposures. We aimed to estimate the comprehensive dose due to internal and external exposures at the particularly high natural radiation area, and to discuss the enhancement mechanism of radon. A car-borne survey was conducted to estimate the external doses from terrestrial radiation. Indoor radon measurements were made in 47 dwellings over three to five months, covering the two typical seasons, to estimate the internal doses. Atmospheric radon gases were simultaneously collected at several heights to evaluate the vertical distribution. The absorbed dose rates in air in the study area vary widely between 50 nGy h-1 and 1109 nGy h-1. Indoor radon concentrations ranged from 124 Bq m-3 to 1015 Bq m-3. That is, the indoor radon concentrations measured exceed the reference levels of 100 Bq m-3 recommended by the World Health Organization. Furthermore, the outdoor radon concentrations measured were comparable to the high indoor radon concentrations. The annual effective dose due to external and internal exposures in the study area was estimated to be 27 mSv using the median values. It was found that many residents are receiving radiation exposure from natural radionuclides over the dose limit for occupational exposure to radiation workers. This enhanced outdoor radon concentration might be as a result of the stable atmospheric conditions generated at an exceptionally low altitude. Our findings suggest that this area provides a unique opportunity to conduct an epidemiological study related to health effects due to chronic low dose-rate radiation exposure.

Isolation and identification of persistent chlorinated organophosphorus flame retardant-degrading bacteria.

Tris(2-chloroethyl) and tris(1,3-dichloro-2-propyl) phosphates are chlorinated persistent flame retardants that have recently emerged as environmental pollutants. Two bacterial strains that can degrade the compounds when they are the sole phosphorus sources have been isolated and identified as members of the sphingomonads. The strains can be useful for the bioremediation of environments contaminated with these compounds.

Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme.

The gene encoding acetylcholinesterase (AChE) was cloned from common carp muscle tissue. The full-length cDNA was 2368 bp that contains a coding region of 1902 bp, corresponding to a protein of 634 amino acids. The deduced amino acid sequence showed a significant homology with those of ichthyic AChEs and several common features among them, including T peptide encoded by exon T in the C-terminus. Three yeast expression vectors were constructed and introduced into the yeast Pichia pastoris. The transformant harboring carp AChE gene lacking exon T most effectively produced AChE activity extracellularly. The replacement of the native signal sequence with the yeast alpha-factor prepro signal sequence rather decreased the production. A decrease in cultivation temperature from 30 to 15 degrees C increased the activity production 32.8-fold. The purified recombinant AChE lacking T peptide, eluted as a single peak with a molecular mass of about 230 kDa on the gel filtration chromatography, exhibited the specific activity of 4970 U/mg. On the SDS-PAGE, three proteins with molecular masses of 73, 54, and 22 kDa were observed. These proteins were N-glycosylated, and their N-terminal sequence showed that the latter two were produced from the former probably by proteolytic cleavage at the C-terminal region. Thus, the recombinant AChE is homotrimer of three identical subunits with 73 kDa. The optimal temperature and pH of the recombinant were comparable to those of the native enzyme purified previously, but the values of kinetic parameters and the sensitivities to substrate inhibition and inhibitors were considerably different between them.

Characteristic of thoron (220Rn) in environment.

This paper describes importance of 220Rn (hereafter thoron) progeny measurement for the dose estimation. Although the spatial distribution of thoron activity concentration strongly depends on the distance from wall surface as an indoor thoron source), a homogeneous distribution was expected to be observed for 212Pb activity concentration which was one of thoron progeny. Furthermore, the mean equilibrium factor for thoron obtained by the recent measurements in several countries widely ranged from 0.008 to 0.07. Therefore the bronchial dose evaluated using the equilibrium factor and activity concentration of thoron instead of thoron progeny activity concentration may have a large uncertainty. Thus, the thoron progeny measurement should be investigated at each measurement point for the dose estimation for thoron.

Measurement system for alpha and beta emitters with continuous air sampling under different exposure situations.

In the present study, a continuous radioactive aerosol measurement system (CRAMS) was developed for measurements of radioactive plume (e.g. 131I, 134Cs and 137Cs) under the emergency situation, and measurements of radon/thoron progeny under the existing situation. As a result, it is suggested that the CRAMS could follow the variation of radon concentration, and the detection limit of the CRAMS under ambient dose equivalent rate of 20µSvh-1, where the temporary evacuation is required within one week in the Japanese regulation, was evaluated to be 129Bqm-3 in the manner of ISO11929.

Cloning and expression of the pyridoxal 5'-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme.

D-aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.

Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme.

The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.