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Multiple epidemiological studies have shown that obesity is a serious risk factor for cancer development. While the underlying mechanisms between obesity and cancer are still unknown, obesity disrupts the role of adipocytes in energy homeostasis, and the alteration of adipokine, insulin and sex steroid signaling. Recently, it has been identified that adipose tissue-derived exosome-like vesicles (ELVs) regulate metabolic homeostasis. In this study, we collected ELVs from adipose tissue of an obese mouse (ob/ob) strain and control mouse (C57BL/6) strain, and checked whether adipose ELVs influence radiation-induced cell death on mouse fibroblast cells (m5S). Furthermore, we analyzed the micronucleus (MN) frequency in survived cells after radiation exposure to investigate the effect of ELVs on radiation-induced genomic instability. We first observed that ELVs from control and obese mice showed enhanced colony forming ability in un-irradiated m5S cells. However, enhanced survival was observed only in 3 Gy-irradiated m5S cells with obese ELV treatment. Despite no ELV effect on colony size, interestingly, the frequency of MN in survived m5S cells after 3 Gy irradiation was elevated when treated obese ELVs compared to control ELVs. These results suggested that obese mouse adipose ELVs could enhance the survival of irradiated cells harboring increased radiation-induced genomic instability.

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract.

The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency.Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 μg/ml) and demecolcine (0.05 μg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures.In WB-culture, MI showed the highest value (∼22%) in 5-15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose ofWhile MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.

Mucus layer that covers the body surface of various animal functions as a defense barrier against microbes, environmental xenobiotics, and predators. Previous studies have reported that L-amino acid oxidase (LAAO), present in several animal fluids, has potent properties against pathogenic bacteria, viruses, and parasites. LAAO catalyzes the oxidative deamination of specific L-amino acids with the generation of hydrogen peroxide and L-amino acid metabolites. Further, the generated hydrogen peroxide is involved in oxidation (direct effect) while the metabolites activate immune responses (indirect effect). Therefore, LAAO exhibits two different mechanisms of bioactivation. Previously, we described the selective, specific, and local oxidative and potent antibacterial actions of various LAAOs as potential therapeutic strategies. In this review, we focus on their biochemical features, enzymatic regulations, and biomedical applications with a view of describing their probable role as biochemical agents and biomarkers for microbial infections, cancer, and autoimmune-mediated diseases. We consider that LAAOs hold implications in biomedicine owing to their antimicrobial activity wherein they can be used in treatment of infectious diseases and as diagnostic biomarkers in the above-mentioned diseased conditions. •Focus on biochemical features, enzymatic regulation, and biomedical applications of LAAOs. •Mechanisms of antimicrobial activity, inflammatory regulation, and immune responses of LAAOs. •Potential biomedical application as an antimicrobial and anti-infection agent, and disease biomarker.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.

L-amino acid oxidases (LAAOs) have antibacterial activity and play important roles in innate immunity. We have previously identified a LAAO of ~52 kDa in size from the mucus layer of the flounder Platichthys stellate (psLAAO1) and have successfully produced psLAAO1 as a secreted bioactive recombinant protein by using Pichia pastoris (P. pastoris). The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in the mucus layer. In this study, homology modeling of psLAAO1 predicted metal coordination by residues Y241, H348, and D406. We show that the Michaelis constant (Km) of psLAAO1 decreased and the catalytic constant (Kcat/Km) value increased following pre-treatment of the protein with a chelating agent. In contrast to the non-chelated protein sample, enzymatic activity of EDTA-treated psLAAO1 gradually decreased or was absent after one or two freeze-thaw cycles. The H348A psLAAO1 mutant generated by site-directed mutagenesis and recombinantly produced by P. pastoris did not display antibacterial activity. The results of the metal detection assay revealed that for the non-metal coordinating histidine mutant (H209A, control), the levels of iron, zinc, and magnesium were similar to those of wild-type psLAAO1, whereas magnesium was not detected in the H348A mutant sample. A wild-type psLAAO1 sample treated with chelating agent did not contain zinc and magnesium ions. In conclusion, metal coordination by psLAAO1 affects enzymatic activity, and H348 is involved in the coordination of magnesium, and metal coordination by psLAAO1 provides essential structural stability. KEY POINTS: • Homology modeling of psLAAO1 predicted metal coordination by residue H348 • The H348A psLAAO1 mutant showed no antibacterial activity or magnesium coordination • Metal coordination by H348 affects enzyme activity and structural stability.

To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 μGy/d (Hirosaki), 14.4 ± 2.4 μGy/d (Tanashio), 208.8 ± 31.2 μGy/d (Ide), 470.4 ± 93.6 μGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones.Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas.Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in Japan on March 11 2011, the surroundings became contaminated with radionuclides. To understand the possible biological effects af...

PURPOSE Cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNC) for dose estimation. Cell-cycle progression parameters of nuclear division index (NDI) and percentage of BNC (% BNC) are also evaluated. Whole blood (WB) or peripheral mononuclear cells (PBMCs) isolated from WB can be used for lymphocyte culture. Previously, 2 Gy PBMCs showed higher NDI and lower MN frequency than WB in 15 ml polypropylene tube single cultures. In this follow-up study, we wanted to assess if soluble factors present in WB but absent in PBMCs could increase MN frequency or decrease NDI in PBMCs co-cultured with WB. MATERIALS AND METHODS Peripheral blood from four healthy donors (two males: 25, 51; two females: 23, 26 years old) was irradiated with X-ray at 1 Gy/min. CBMN assay was performed with different combinations of 0 and 2 Gy WB and PBMC (WB, WB-IR, PBMC, PBMC-IR) mono- and co-cultures in a polystyrene six-well plate. Co-cultures were separated by 0.4 µm transwell inserts. Log2 fold changes and values of NDI, % BNC and MN frequency analyzed by three scorers were obtained. RESULTS As upper and lower wells of the same culture condition showed some significant differences, wells of the same level were compared. NDI of PBMCs increased when PBMC or PBMC-IR was co-cultured with WB or WB-IR, respectively, as compared to mono-cultures. There was no increase in PBMC-IR's MN frequency when co-cultured with WB or WB-IR. MN frequency was consistently higher in WB-IR than PBMC-IR in both mono- and co-cultures. NDI, % BNC and MN frequency were similar when WB or PBMC were co-cultured with PBMC-IR or WB-IR, respectively. Significantly lower NDI and % BNC, and higher MN frequency were also seen in some conditions of 15 ml cultures than six-well mono-cultures. CONCLUSIONS Instead of the hypothesized decrease in NDI and increase in MN frequency, our co-culture set-up showed that in the absence of direct cell-cell interaction, soluble factors in WB increased NDI but not MN frequency in PBMCs. Moreover, radiation-induced bystander effects could not be observed. As the type of cell culture (WB, PBMC) and culture vessels could influence NDI and MN frequency, CBMN culture protocols should be kept consistent for dose-response calibration curve construction and dose estimation.

Hirosaki University has been designated by the Nuclear Regulation Authority as an Advanced Radiation Emergency Medical Support Center and as a hospital which accepts radiation emergency medical patients in Japan. One of the efforts of the Advanced Radiation Emergency Medicine Support Center is cytogenetic biodosimetry in radiation emergency medicine. We are working on validating all the steps from blood collection and storage to DNA damage evaluation. We are also improving various methodologies, such as the cytokinesis-block micronucleus (CBMN) assay. In our improved CBMN harvest protocol to store fixed isolated PBMCs (peripheral blood mononuclear cells), a high frequency of scorable binucleated cells can be obtained with routine biodosimetry reagents and equipment. In addition, we are strengthening infection control measures and working to establish a system that enables safe and reliable dose estimation of exposed patients during the COVID-19 pandemic. Furthermore, we have developed on-campus education system for undergraduate and graduate students, and programs for off-campus trainees, such as "International Training Program for Radiation Protection" to develop human resources responsible for cytogenetic biodosimetry. In this time, I would like to introduce the efforts of the cytogenetic biodosimetry laboratory in Hirosaki University, Japan.

In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods.Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy - background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated.94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures.This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident in Japan on March 11 2011, the surroundings became contaminated with radionuclides. To understand the possible biological effects after chronic low dose-rate radiation in contaminated areas of Fukushima, we assessed the effects in large Japanese field mice (We collectedAlthough the mice in the contaminated areas were chronically exposed, there was no radiation-specific chromosome aberrations observed, such as dicentric chromosomes and rings. Some structural aberrations such as gaps and breaks were observed, and these frequencies decreased annually in mice from Namie Town.These findings suggest that chromosome aberration analysis is useful to evaluate and monitor radiation effects in wild animals.

In order to establish suitable protocols of blood culture to obtain sufficient numbers of metaphases for dicentric chromosome assay (DCA), we have examined the effect of storage temperature, storage time, and anticoagulant type.Peripheral blood was collected from five healthy donors with lithium heparin and ethylenediaminetetraacetic acid dipotassium salt (EDTA-2K). These samples were irradiated with X-rays at 3 Gy or sham; the samples were further divided into groups that were either stored at room temperature (RT) or 5.2 ± 1.0 °C. After 6, 24, 48, 72, and 168 h of storage, both blood counts and the mitotic index (MI) were analyzed.Heparinized blood samples stored under cold conditions exhibited low white blood cell, lymphocyte, and platelet counts. EDTA-treated blood samples did not show such obvious changes in cell counts. After 6 h of storage, heparinized blood samples stored at RT had MI of 21.5-29.3%. Similar MI was obtained in the EDTA-washed group stored for 6, 24, 48, and 72 h.Our study confirms that heparinized blood samples should be stored at RT to get sufficient metaphases for DCA, and that EDTA blood samples also can be used for blood culture after washing and storage under 5.2 ± 1.0 °C.

Balamuthia mandrillaris is a free-living amoeba that lives in soil and water near human settlements. B. mandrillaris was first isolated from a mandrill baboon that died at the San Diego Zoo Wildlife Park in California in 1986, and the first human infection was reported in 1990. Although reported B. mandrillaris infections are often not properly characterized, it appears that B. mandrillaris invades the living body from the soil and water, either via a wound or the nasal cavity. Most confirmed infections have originated in South and North America. B. mandrillaris inhabits warm climates and is recognized as a pathogen in warm areas such as desert climates and tropical climates. B. mandrillaris has been isolated from environmental samples since 2000, most of which originated from warm areas such as step climates, tropical climates, and desert climates. However, B. mandrillaris may survive in diverse environments, although fewer granulomatous amebic encephalitis patients have been reported in colder Japanese and Northern European regions. In the present study, we conducted a survey of 13 soil samples in Aomori Prefecture located at the northernmost tip of Japan Honshu and successfully isolated one strain of B. mandrillaris from soil for the first time in Japan. In addition, B. mandrillaris gene was detected from several soils. This confirms that B. mandrillaris is capable of spreading to a wider climatic region.

Although evidence suggests that ionizing radiation can induce the bystander effect (radiation-induced bystander effect: RIBE) in cultured cells or mouse models, it is unclear whether the effect occurs in cells of wild animals. We investigated medium-mediated bystander micronucleus (MN) formation and DNA damage in un-irradiated cells from a large Japanese field mouse (Apodemus speciosus). We isolated four clones of A. speciosus embryonic fibroblasts (A603-1, A603-2, A603-3, and A603-4) derived from the same mother, and examined their radiation sensitivity using the colony-forming assay. A603-3 and A603-4 were similar, and A603-1 and A603-2 were highly sensitive compared with A603-3 and A603-4. We examined RIBE in the four clones in autologous medium from cell cultures exposed to 2 Gy X-ray radiation (irradiated cell conditioned medium: ICCM). We only observed increased MN prevalence and induction of DNA damage foci in A603-1 and A603-3 cells after ICCM transfer. The ICCM of A603-3 (RIBE-induced) was able to induce MN in A603-4 (not RIBE-induced). To assess the possible contribution of reactive oxygen species (ROS) or nitric oxide (NO) in medium-mediated RIBE, dimethyl sulfoxide (DMSO; a ROS scavenger) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; an NO scavenger) were added to the medium. A suppressive effect was observed after adding DMSO, but there was no effect after treatment with c-PTIO. These results suggest that an enhanced radiosensitivity may not be directly related to the induction of medium-mediated RIBE. Moreover, ROS are involved in the transduction of the RIBE signal in A. speciosus cells, but NO is not. In conclusion, our results suggest that RIBE may be conserved in wild animals. The results contribute to better knowledge of radiation effects on wild, non-human species.

Primary hepatocyte culture is a crucial tool for investigations of liver function and for evaluating the toxic effects of drugs. In addition, chromosomal analysis of hepatocytes could also prove useful for understanding the mechanisms of hepatocarcinogenesis. However, cultivation of primary hepatocytes for chromosome analysis has been hampered by the specific equipment and skill required to perform the in situ perfusion step necessary for isolation of primary hepatocytes. In the present study, we aimed to establish a simple and efficient method of isolating hepatocytes suitable for chromosome analysis. We performed hepatocyte isolation without using collagenase perfusion, instead digesting liver tissues using collagenase in tubes. In addition, we examined hepatocyte and bone marrow cell (BMC) co-culture and cultivation of hepatocytes with medium containing BMC culture medium supernatants. We found that hepatocyte viability and attachment rate were significantly improved, both by co-culture with BMCs and medium containing BMC culture media supernatants, with the latter also significantly increasing the mitotic index. Using this simple method of isolation and cultivation, we could successfully perform chromosomal analysis of mouse primary hepatocytes. This method has the potential to help understand the mechanisms underlying chromosomal instability-mediated hepatocarcinogenesis.

Age at exposure is a critical factor that influences the risk of radiation-induced leukemia. Accumulating evidence suggests that ionizing radiation can induce genomic instability and promote leukemogenesis in hematopoietic stem cells (HSCs); however, the influence of age on this phenomenon has not been elucidated. In this study, infant (1-week-old) or adult (14-week-old) C3H/He mice received sham or 4 Gy whole-body irradiation, and bone marrow cells were transplanted to recipients at day 1 or 60 postirradiation. Twelve days after bone marrow transplant, we analyzed the radiation-induced genomic instability by scoring the frequency of DNA damage and micronucleus formation in colony-forming units-spleen (CFU-Ss). We observed significant increases in DNA damage and micronucleus formation in CFU-Ss of the 4 Gy irradiated adult cells transplanted at day 1 or 60 postirradiation. However, the frequency of DNA damage focus and micronucleus formation in CFU-Ss of 4 Gy irradiated infant cells transplanted at day 1 or 60 postirradiation was relatively decreased. Quantitative differences in the reactive oxygen species and cells expressing inducible nitric oxide synthase in CFU-Ss suggested that age-dependent radiation-induced genomic instability may result from chronic oxidative stress by pro-inflammatory states in HSC descendants after radiation exposure.

Introduction:Due to the heatwave in Japan, the number of patients with heatstroke transported via ambulance hit the highest record according to the Tokyo fire department in 2018. Now, heatwaves are thought to be a natural disaster. Emergency medical professionals located in a Tokyo suburb covering 40,000 individuals experienced a wide range of challenges in patient care with heatstroke.Aim:To assess the impact of the 2018 Japan heatwave on community emergency medical service.Methods:Patients (aged ≥ 16 years) with heatstroke and who were transported to our hospital by ambulance from June to September of 2018 were included. Data were derived from prehospital records and electronic medical records. Weather data was referenced from a Japanese meteorological agency.Results:The number of all-cause cases was 1,764, and the total number of heatstroke cases was 51 (2.9%). Heatstroke cases were concentrated in July and August of 2018. The rate of males was 69%. The average age was 63 ± 23 years. Physical labor was associated with 31% of cases, such as sports in 7.8%. Hospitalization was required in 24%. ICU admission was required in 9.8%. There was no fetal case directly caused by heatstroke in this survey.Discussion:The risk factors of heatstroke considered to be male and elderly. About one-half of heatstroke patients were over 70 years old, and it may have been related to regional characteristics. As mentioned in the Heatstroke STUDY 2012 (Miyake, 2014), most cases which occurred inside residences were found that there was no air conditioner use. There was also an increased number of patients with heatstroke who made emergency visits by themselves. Further investigation is needed annually to estimate the effect of climate change. It is important to make a strong recommendation from public health agency about heatstroke prevention, including air conditioner use during hot weather.

Venom, the mucus layer covering the body surface, ink glands, mammary glands, milk, and various animal secretory functions as both a physical and chemical defense barrier against bacteria and virus infections. Previously, several studies reported that L-amino acid oxidases (LAAOs) present in animal secretary fluids have strong antimicrobial activities and selective cytotoxic activities against Gram-positive and Gram-negative bacteria, various pathogenic bacteria, viruses, and parasite species. These LAAOs catalyze oxidative deamination of an L-amino acid substrate with the generation of hydrogen peroxide. The antibacterial activity of LAAOs is completely inhibited by catalase; thus, LAAOs kill bacteria by the hydrogen peroxide generated from the oxidation of L-amino acid substrates. This review focuses on the selective, specific, and local antibacterial actions of various LAAOs that may be used as novel therapeutic agents against infectious diseases. LAAOs that are suitable leads for combating multidrug-resistant bacterial infections are also studied.

Objective: This study aims to investigate the effects of surface bacteria of buckwheat sprouts on obesity. We examine whether these bacteria have probiotic properties. Methods: Since P-36 and P-37 strains were present in large amounts on the buckwheat sprouts surface, identification of species was carried out by intestinal bacteria identification kit and 16S rRNA gene analysis. Subsequently, to investigate the biological effects, P-36 and P-37 strains were divided into two groups, heat-treated dead bacteria and untreated viable bacteria, which were then orally administered to obese model mice exhibiting hyperglycemia every other day for a total of 10 times. After administration, BMI and visceral fat mass were measured, and oral glucose tolerance test was conducted to evaluate glucose tolerance; their influence on blood glucose level and insulin resistance (HOMA-IR) was evaluated. Results: The strain P-36 was identified as Pantoea sp., and P-37 as Kosakonia cowanii. In the group of mice administered the living and dead bacteria of P-36 and P-37, the weight gain rate after the administration was low, and the progression of obesity was suppressed. In the oral glucose tolerance test, an improvement of glucose tolerance and a significant suppression of blood glucose level was confirmed at 0 and 15 min in the group treated with viable P-37. In addition, HOMA-IR was improved in mice treated with both viable and dead P-37 bacteria. Conclusion: The mice administered P-36 and P-37 showed a gradual increase in body weight and a decrease in visceral fat percentage. The mechanism leading to improved blood glucose levels observed in mice administered P-37 strain remains unclear at present. The involvement of buckwheat sprouts-derived bacteria in improving hyperglycemia and reducing obesity during a short time will help to discover new probiotics.

Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial l-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane.

The calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method for assessing the cell-cycle distribution in cells, since calyculin A induces chromosome condensation in various phases of the cell cycle. In this study, a novel parameter, the cell-cycle progression index (CPI), in the PCC assay was validated as a novel biomarker for biodosimetry. Peripheral blood was drawn from healthy donors after informed consent was obtained. CPI was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60)Co-gamma rays: ∼0.6 Gy min(-1), or X ray: 1.0 Gy min(-1); 0-10 Gy) model. The calyculin A-induced PCC assay was performed for chromosome preparation. PCC cells were divided into the following five categories according to cell-cycle stage: non-PCC, G1-PCC, S-PCC, G2/M-PCC and M/A-PCC cells. CPI was calculated as the ratio of G2/M-PCC cells to G1-PCC cells. The PCC-stage distribution varied markedly with irradiation doses. The G1-PCC cell fraction was significantly reduced, and the G2/M-PCC cell fraction increased, in 10-Gy-irradiated PBL after 48 h of culture. CPI levels were fitted to an exponential dose-response curve with gamma-ray irradiation [y = 0.6729 + 0.3934 exp(0.5685D), r = 1.0000, p < 0.0001] and X-ray irradiation [y = -0.3743 + 0.9744 exp(0.3321D), r = 0.9999, p < 0.0001]. There were no significant individual (p = 0.853) or gender effects (p = 0.951) on the CPI in the human peripheral blood ex vivo irradiation model. Furthermore, CPI measurements are rapid (< 15 min per case). These results suggest that the CPI is a useful screening tool for the assessment of radiation doses received ranging from 0 to 10 Gy in radiation exposure early after a radiation event, especially after a mass-casualty radiological incident.

Background P2Y purinergic receptors (P2YR) are G protein-coupled receptors that are stimulated by extracellular nucleotides. They mediate cellular effects by regulating cAMP production, protein kinase C activation, inositol trisphosphate generation, and Ca 2+ release from intracellular stores. The P2Y6 receptor of this family is selectively stimulated by UDP, and selectively inhibited by MRS2578. In the present study, we examined the effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in human basophils. Methods Basophils were purified from human peripheral blood. The mRNA expression of genes encoding P2YR and ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) was measured by RT-PCR. Intracellular Ca 2+ influx via UDP/P2Y6 receptor signaling in basophils was detected using a calcium probe. The effect of UDP/P2Y6 receptor signaling on IgE-dependent degranulation in basophils was confirmed by measuring CD63 expression by flow cytometry. Autocrine secretion of nucleotides was detected by HPLC analysis. Results We showed that purified basophils express P2Y6 mRNA and that UDP increased intracellular Ca 2+ , which was reduced by MRS2578 treatment. UDP promoted IgE-dependent degranulation. Furthermore, MRS2578 inhibited IgE-dependent degranulation in basophils. HPLC analysis indicated that basophils spontaneously secrete UTP. In addition, basophils expressed the extracellular nucleotide hydrolases ENTPDase2, ENTPDase3, and ENTPDase8. Conclusions This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

We investigated the process of induction of preneoplastic cells positive for glutathione S‐transferase P‐form (GST‐P) in the rat liver. AAF (2‐Acetylaminofluorene) mixed with normal rat chow at high concentration (0.04%) induced 517 000 ± 86 000 GST‐P (+) single hepatocytes/g liver after 2 weeks followed by induction of a few foci and nodules after 4–6 weeks. Overproduction of GST‐P (+) single hepatocytes was dose‐ and time‐dependent, and the induction kinetics were typical of first‐order consecutive reaction, by which induction of the positive cells was nongenetic. Quantitative analysis indicated that the estimated numbers of cells in foci and nodules at 4–6 weeks after exposure to AAF ranged from 2.7 × 10(4) (2(14.7)) to 3.6 × 10(6) (2(21.7)) cells, and 2.0 × 10(4) (2(14.3)) to 2.7 × 10(6) (2(21.4)) cells, respectively, when analyzed by using two equations. According to the initiated cell theory of Farber, foci and nodules are formed through sequential cell division of 14 to 21‐times or more within a short time period. The rapid growth exceeded the rate of cell division, indicating that the growth of preneoplastic cells is based on a nonclonal penetration mechanism. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02325.x, 2012)

Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.

We present a case of hepatoid carcinoma of the abdominal skin in a male Wistar rat. Histopathologically, this carcinoma resembled human hepatocellular carcinoma with respect to trabecular-sinusoidal structures. Carcinoma tissues contain numerous eosinophilic globules and crystals, and in this case, we found the characteristic eosinophilic globules in the hepatoid carcinoma cells and the crystals in the extracellular portions. Vivid carcinoma cells full of eosinophilic globules were present near the necrotic areas in tumor tissue, wherein quadrate crystals unstained with eosin were observed. PAS staining after diastase digestion revealed that the globules were PAS positive and diastase resistant. In addition, we found that the hepatoid carcinoma cells were immunoreactive for alpha-1-antitrypsin (anti-A1AT) antibody with the globules and crystals staining peripherally, and a central unstained region. Ultrastructural study of intracytoplasmic globules and extracellular crystals revealed that the fringe of each globule and crystal had no limiting membrane and showed the same level of electron density. These findings suggest that the characteristic crystals in this tumor may have originated from the globules that were emitted from the carcinoma cells after their death as a result of saturation with intracytoplasmic globules.

Respiratory dysfunction remains one of the major complications after burn surgery in extensively burned patients. We evaluated the relationship between the invasiveness of burn surgery and acute lung injury (ALI).Patients admitted to our burn unit between 2006 and 2007 with burns greater than or equal to 30% of the total body surface area without severe inhalation injury were entered into this study. Of sixteen patients (mean age, 49.4 +/- 19.3 years, total body surface area 46.0% +/- 14.9%) who underwent burn surgery (3-6 days postburn), vital signs, hemodynamic parameters, blood gas analysis, and peak inspiratory pressure were recorded. Lung injury severity (LIS) score was serially determined. Bronchoalveolar lavage (BAL) was performed before and 24 hour postoperatively.Body temperature and LIS score preoperatively (baseline) were 37.1 degrees C +/- 0.9 degrees C and 3.8 degrees C +/- 2.2 degrees C, respectively. LIS score increased with increased polymorphonuclear neutrophil in BAL 24 hour postoperatively in 7 of 10 patients with intraoperative temperature decreasing more than 1 degrees C. Extent of excision (20.3% +/- 6.7%), transfusion (4.3 +/- 3.0 units), or duration of surgery (147 +/- 49 minutes) alone did not show significant correlation with the development of ALI postoperatively.In patients with severe burn injury, hypothermia during surgery despite aggressive intraoperative warming is significantly correlated with the development of ALI with increased polymorphonuclear neutrophil in BAL and may reflect the severity of invasiveness.

Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

Characteristics of the cellular response to oxygen deprivation and subsequent reoxygenation (hypoxia/reoxygenation) include redirection of energy metabolism, increased glucose utilization and expression of oxygen-regulated proteins. Inhibition of protein synthesis during early reoxygenation period prevented effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death. To elucidate the role of astrocytes in the central nervous system in response to hypoxia/reoxygenation, we analyzed the cDNA library derived from the cultured rat astrocytes subjected to 24 h of hypoxia followed by reoxygenation by differential display, and isolated a cDNA corresponding to Na/K ATPase alpha1 subunit. The expression of Na/K ATPase alpha1 subunit mRNA as well as beta1subunit mRNA was transiently increased after reoxygenation, whereas hypoxia itself did not induce any gene expression change. Na/K ATPase alpha1 subunit protein was transiently increased, whereas the protein expression for Na/K ATPase beta1 subunit showed sustained induction after reoxygenation. Overexpression of beta1 subunit in HEK 293 cells subjected to hypoxia/reoxygenation promoted survival of the cells. These findings suggest that Na/K ATPases may contribute to maintain the cellular environment of astrocytes subjected to hypoxia/reoxygenation.

切除不能な乳癌肝転移に対して間欠的肝動注を行ったところ著効し, その後に肝切除を施行できた症例を経験したので報告する.症例は61歳の女性. 1994年8月頃から左乳房の腫瘤に気づき, 急速に増大したため当科へ紹介された. 1995年2月14日, 左乳癌の診断で胸筋合併乳房切除術を施行した. 同年10月, 肝両葉に多数の転移をきたしたため, 5-fluorouracil 500mgとcarboplatin 100mgの間欠的肝動注を開始した. 肝腫瘍は徐々に縮小し, 14か月後には病変が右葉に限局したため, 1997年1月14日, 肝右葉切除術を施行した.病理組織学的に肝病変は線維化を伴った壊死組織のみで腫瘍細胞を認めず, complete responseの状態であった. 一時, tumor freeとなったが, 同年6月に肺転移および脳転移が出現し, 両者に対して放射線照射および全身化学療法を施行したが, 1998年4月28日に呼吸不全のため死亡した.

The effect of 4-methylumbelliferone (MU), a hyaluronan synthase-suppressor, on O-linked β-Nacetylglucosaminylation (O-GlcNAcylation) was investigated in cultured human skin fibroblasts, and we found that MU stimulated O-GlcNAcylation of the cellular proteins. Since O-GlcNAcylation affects protein phosphorylation via Ser/Thr kinases, we examined the effect of MU on both the phosphorylation of hyaluronan synthase 2 (HAS2) and hyaluronan production. The cells were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and MU independently or in combination. The protein fraction of each cell culture was extracted and divided into 2 parts-phosphorylated and non-phosphorylated fractions-by immobilized metal-affinity chromatography. The hyaluronan level in the medium was determined by an ELISA-like assay. Addition of MU decreased the level of hyaluronan in the medium and that of HAS2 in the phosphorylated protein fraction. On the contrary, the addition of TPA increased the levels of both of them. Interestingly, the combination of TPA and MU lowered the levels of them in treated cells as compared to those in untreated control cells. These results suggest that TPA activated protein kinase C (PKC), which stimulates the phosphorylation of HAS2, and increased hyaluronan production. Further, MU may inhibit the phosphorylation of HAS2 by PKC through the stimulation of O-GlcNAcylation.

Ionizing radiation (IR) causes DNA injury and induces multiple signal mechanisms, including the regulation of DNA repair, the cell cycle and gene expression through the activation of p53-related pathways. Cis-natural antisense transcripts (cis-NATs), which are transcribed from the DNA strand opposite to that for mRNA of the gene, are recognized as important regulators of gene expression in eukaryotic cells, but the effects on cis-NAT expression by IR are unknown to date. Therefore, we investigated the effects of X-ray irradiation on cis-NAT expression together with mRNA expression using a human B lymphoblast cell line (IM-9), a custom-microarray and strand-specific RT-qPCR. Eighteen, 33 and 106 mRNAs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (fold change, FC >2.0). On the other hand, 10, 22 and 43 NATs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (FC >2.0). Among these mRNAs/NATs, the IR dose-dependent up-regulation of mRNAs and cis-NATs of MDM2 and CDKN1A were confirmed by strand-specific RT-qPCR. Additionally, the cis-NATs of MDM2 were indicated to be localized in the cytoplasm, while cis-NATs of CDKN1A were located in the nucleus and cytoplasm. In conclusion, the radiation-responsive cis-NATs in conjunction with mRNAs were identified for the first time in the present study. It is possible that these cis-NATs regulate the gene expression in a post-transcriptional fashion. The IR dose-dependent up- and down-regulation of these mRNAs/cis-NATs may be a marker for ionizing radiation.

Aims The aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix. Main methods After obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24–48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting. Key findings LPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each. Significance This study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.

Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.

Secondary abdominal compartment syndrome (ACS) is a lethal complication after resuscitation from burn shock, even after abdominal decompression (AD) is performed. This study investigated increased susceptibility to multiple organ dysfunction syndrome (MODS) in extensively burned patients with ACS.Patients admitted to our burn unit between 2002 and 2005 with burns affecting 40% or more of the total body surface area without severe inhalation injury were analyzed. Hemodynamic parameters, blood gas analysis, and intrabladder pressure as intra-abdominal pressure were recorded. Serum interleukin (IL)-8 and IL-6 concentrations were measured in 20 of these patients. Lung injury score and Sequential Organ Failure Assessment scores were serially determined.Fourteen of 38 patients developed intra-abdominal hypertension in 22.9 +/- 8.9 hours postburn. Hemodynamic parameters in these 14 patients, including peak intra-abdominal pressure (46.6 +/- 11.2 to 19.8 +/- 9.9 cm H2O), peak inspiratory pressure (51.4 +/- 10.5 to 31.8 +/- 7.0 cm H2O), and abdominal perfusion pressure (51.3 +/- 18.3 to 73.9 +/- 13.6 mm Hg), were improved immediately after AD. Despite AD, lung injury score and Sequential Organ Failure Assessment scores increased significantly 2 and 3 days postburn in patients who required AD. Plasma concentration of IL-8 was elevated in intra-abdominal hypertension patients 3 days postburn.Intra-abdominal hypertension induced acute lung injury and MODS with IL-8 elevation, even though AD improved hemodynamic parameters in extensively burned patients.

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. Here, we report a candidate of the I gene, GmIRCHS, which consists of a 5'-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5'-DeltaCHS3 and 3'-DeltaCHS3). RT-PCRs and RNase protection assay indicated the existence of the read-through product from 5'-DeltaCHS3 to 3'-DeltaCHS3 and the dsRNA region of DeltaCHS3, suggesting that dsRNA of DeltaCHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of DeltaCHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene.

The P2Y receptors (P2YRs) are a family of G protein-coupled purinergic receptors that are stimulated by ATP, ADP, UTP, UDP, and UDP-G, and their interaction induces a wide range of biological effects in various cells through phospholipase C activation, inositol triphosphate generation, and Ca2+ release from intracellular stores. In our previous study, we demonstrated that in neutrophils, ATP regulates chemotaxis and phagocytosis via P2YR. In the present study, we examined the distribution and function of P2YR in another innate immune cell type, basophils, the degranulation of which is associated with type I allergy. To date, P2YR have been subdivided into 8 different subclasses: P2Y1, 2, 4, 6, 11, 12, 13, and 14. Of these 8 subclasses, our RT-PCR results showed the predominant expression of P2Y2, P2Y4, and particularly P2Y6, which are known as uracil nucleotide-sensitive P2YRs. Interestingly, HPLC analysis indicated that basophils spontaneously secrete UTP, the ligand for P2Y2 and 4, whereas UDP, the ligand for P2Y6, is probably generated from UTP in a metabolic pathway. Although the addition of UTP and UDP did not directly induce basophil degranulation, these nucleotides clearly enhanced the degranulation induced by anti-IgE cross-linking. These results suggest that uracil nucleotides act as potent inducers of histamine release in basophils.