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A diesel exhaust particle (DEP) is a type of particulate matter that is easily produced from combustion in a diesel power engine. It has been reported that DEPs can cause short- and long-term health problems. This is because DEPs are complex mixtures that are highly inhalable through the airways due to their small particle size. However, the relationship between intracellular localization of DEPs after their deposition in the lungs and the subsequent biological responses remains to be clarified. This is due to difficulties in distinguishing particles that are inside the cells from those that are outside. In this study, A549 human lung epithelial cells were exposed to DEPs at concentrations of 0, 25, 75, or 200 µg/mL for different periods, after that particles in the A549 cells were analyzed by three-dimensional (3D) images obtained from a Raman microscope. The cytotoxic effects of DEPs on the A549 cells were investigated by measuring cell viability, the levels of intracellular reactive oxygen species (ROS) and cell death. The Raman microscopy revealed that the particles invaded the A549 cells, and at a concentration of 200 µg/mL, they markedly decreased cell viability, increased intracellular ROS production, triggered late apoptosis/necrosis and induced nuclear damage. These results suggest that intracellular DEPs exposed at a high concentration may be highly toxic and can impair the viability of A549 cells. Furthermore, the 3D images from the Raman microscopy can be used to evaluate intracellular particle dynamics.

Epidemiological and experimental studies have shown that di-(2-ethylhexyl) phthalate (DEHP), a plasticizer, can aggravate allergic diseases. DEHP promotes adaptive immune responses, although its effect on the innate immune system remains largely unknown. The present study investigated the effects of DEHP on group 2 innate lymphoid cells (ILC2) that produce Th2 cytokines in response to epithelial cell-derived cytokines, such as interleukin (IL)-33. ILC2 (lineage-negative, CD45.2+, Sca1+, KLRG1+) were isolated from the lungs of C57BL/6 J mice. Co-exposure to DEHP and IL-33 significantly increased IL-5 release from ILC2, whose level was higher than that of the vehicle and IL-33 alone. The effects of DEHP in the presence of IL-33 showed an inverted-U dose-response. The present is the first report showing that DEHP exacerbates allergy through the innate immune system.

Recent studies have shown that air pollution is intense and hazardous in Asia compared to other parts of the world due to the late and poor implementation of updated technology in automobiles and industry as well as to the high population density. Respiratory disease, including asthma, is exacerbated by air pollution. However, the effects of PM_(2.5), especially on respiratory allergies in Asian cities, have not yet been examined in detail. In this study, airway epithelial cells were exposed to crude PM_(2.5) particles collected by cyclonic separation from three different Asian cities, namely, Sakai, Bangkok, and Taipei. We compared the cytotoxicity and inflammatory potential of the PM_(2.5) from these cities by measuring IL-6 and IL-8. The samples from Sakai and Bangkok caused cytotoxic effects at a dose of 75 μg mL^(-1) and, moreover, induced the release of IL-6 and IL-8 even at low doses. The release of these two interleukins was highly associated with fluoranthene derivatives, microbial factors (endotoxin and β-glucan), metals (e.g., Ti), and organic (OC2 and OC3) and elemental carbon (EC1) in the PM_(2.5). Thus, these components potentially contribute to cellular damage and a pro-inflammatory response in the airway epithelial cells, and the effect depends on PM_(2.5) sources in the locations.

Measures for protecting against PM2.5 (particulate matter with aerodynamic diameters ≤2.5 µm), which exacerbates respiratory diseases, have not been established. The present study investigated the effects of extracts of curry powder and its components on pro-inflammatory responses, extracellular and intracellular reactive oxygen species (ROS) production, induced by the PM2.5 component, DEP (diesel exhaust particles). Airway epithelial cells were exposed to DEP in the presence of curry powder, or its major and/or anti-inflammatory components, clove and turmeric. Curry powder, clove, and turmeric inhibited DEP-induced IL-6 release and extracellular ROS; in the absence of clove and turmeric, these effects of curry powder were mild but similar. Among the other curry spices, cinnamon decreased IL-6 and extracellular ROS, and coriander decreased IL-6 alone. This is the first report on the protective effects of extracts of curry powder and its components, against PM2.5-induced airway inflammation, which may be partly through inhibition of extracellular ROS.

Background Kidney dysfunction is considered a cardiovascular risk factor. However, few longitudinal studies have examined the effects of air pollution on kidney function. We evaluated associations between long-term air pollution exposure and estimated glomerular filtration rate (eGFR) using data from a cohort of the Electricity Generating Authority of Thailand (EGAT) study in Bangkok Metropolitan Region, Thailand. Methods This longitudinal study included 1839 subjects (aged 52–71 years in 2002) from the EGAT1 cohort study during 2002–2012. eGFR, based on creatinine, was measured in 2002, 2007, and 2012. Annual mean concentrations of air pollutants (i.e., particulate matter with an aerodynamic diameter ≤10 μm (PM10), ozone (O3), nitrogen dioxide (NO2), sulfur dioxide (SO2), and carbon monoxide (CO)) prior to a measurement of creatinine were assessed with the ordinary kriging method. Mixed-effect linear regression models were used to assess associations between air pollutants and eGFR, while controlling for potential covariates. eGFR values are expressed as percent change per interquartile range (IQR) increments of each pollutant. Results Lower eGFR was associated with higher concentrations of PM10 (−1.99%, 95% confidence interval (CI): −3.33, −0.63), SO2 (−4.89%, 95%CI: −6.69, −3.07), and CO (−0.97%, 95%CI: −1.96, 0.03). However, after adjusting for temperature, relative humidity, PM10, and SO2, no significant association was observed between CO and eGFR. Conclusions Our findings support the hypothesis that long-term exposure to high concentrations of PM10 and SO2 is associated with the progression of kidney dysfunction in subjects of the EGAT cohort study.

Ambient particulate matter (PM) epidemiologically exacerbates respiratory and immune health, including allergic rhinitis (AR) and bronchial asthma (BA). Although fine and coarse particles can affect respiratory tract, the differences in their effects on the upper and lower respiratory tract and immune system, their underlying mechanism, and the components responsible for the adverse health effects have not been yet completely elucidated. In this study, ambient fine and coarse particles were collected at three different locations in Japan by cyclone technique. Both particles collected at all locations decreased the viability of nasal epithelial cells and antigen presenting cells (APCs), increased the production of IL-6, IL-8, and IL-1β from bronchial epithelial cells and APCs, and induced expression of dendritic and epithelial cell (DEC) 205 on APCs. Differences in inflammatory responses, but not in cytotoxicity, were shown between both particles, and among three locations. Some components such as Ti, Co, Zn, Pb, As, OC (organic carbon) and EC (elemental carbon) showed significant correlations to inflammatory responses or cytotoxicity. These results suggest that ambient fine and coarse particles differently affect nasal and bronchial epithelial cells and immune response, which may depend on particles size diameter, chemical composition and source related particles types.

Particulate matter with diameters

Evaluation of the health effects of particulate matter with aerodynamic dias. ≤ 2.5 μm (PM2.5) should reflect realistic condition in ambient atmosphere. However, using conventional filtration methods, only extracts from PM2.5 collected on the filter can be analyzed and not the particle itself. Cyclonic separation is a technique that enables the direct analysis of the effects of the crude “powder form” of PM2.5 on respiratory health. Airway epithelial cells and antigen-presenting cells were exposed to PM2.5 collected during the same period using a conventional filtration method or cyclonic separation. PM2.5 collected using cyclonic separation led to a higher secretion of interleukins 6 and 8 (IL-6, IL-8) from airway epithelial cells, and IL-6, IL-1β, tumor necrosis factor-α (TNF-α) secretion, cluster of differentiation 86 (CD86), and dendritic and epithelial cells 205 (DEC205) expression on antigen-presenting cells, compared with the effects of filter-collected PM2.5. Furthermore, PM2.5 collected using cyclonic separation increased inflammatory cytokine levels and induced lung inflammation in vivo. These results suggest that crude PM2.5 collected using cyclonic separation causes stronger biological responses than filter-collected PM2.5. Hence, PM2.5 collected using cyclonic separation can be utilized for a reliable evaluation of the health effects of ambient PM2.5.

We investigated the synergic effects of components of particulate matter with aerodynamic diameters ≤2.5 μm (PM2.5) on airway inflammation. Co-exposure to cadmium (Cd) and 9,10-phenanthrenequinone (9,10-PQ) additively/synergistically increased pro-inflammatory responses in airway epithelial cells, whereas co-exposure to Cd and phenanthrene resulted in no acceleration. These results suggest that the combination of metal and a quinone derivative can contribute to the exacerbation of respiratory diseases by PM2.5.

Particulate matter with aerodynamic diameter ≤2.5 μm (PM2.5 ) is generally composed of carbon nuclei associated with various organic carbons, metals, ions and biological materials. Among these components, polyaromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP) and quinones have detrimental effects on airway epithelial cells and immunodisrupting effects, which leads to the exacerbation of respiratory allergies. The effects of PAHs and the carbon nuclei, separately as well as in combination, remain to be established. We investigated the effects of BaP, 9,10-phenanthroquinone (9,10-PQ), and 1,2-napthoquinone (1,2-NQ) and their combined effects with heated diesel exhaust particle (H-DEP) as carbon nuclei of typical PM2.5 . We exposed human airway epithelial cells (BEAS-2B), murine bone marrow-derived antigen-presenting cells (APCs), and murine splenocytes to BaP, 9,10-PQ, or 1,2-NQ in the presence and absence of H-DEP. Several important inflammatory cytokines and cell surface molecules were measured. PAHs alone did not have apparent cytotoxic effects on BEAS-2B, whereas combined exposure with H-DEP induced noticeable detrimental effects which mainly reflected the action of H-DEP itself. BaP increased CD86 expression as an APC surface molecule regardless of the presence or absence of H-DEP. None of the BaP, 9,10-PQ, or 1,2-NQ exposure alone or their combined exposure with H-DEP resulted in any significant activation of splenocytes. These results suggest that PAHs and carbon nuclei show additive effects, and that BaP with the carbon nuclei may contribute to exacerbations of allergic respiratory diseases including asthma by PM2.5 , especially via antigen-presenting cell activation.

Epidemiologic studies have revealed that Asian sand dust particles (ASDs) can affect respiratory and immune health represented by asthma. Factors responsible for the exacerbation of asthma remain unclear. The fungus Bjerkandera adusta (B.ad) and polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) have been identified in ASDs collected from the atmosphere when an ASD event occurred. We investigated the effects of B.ad and BaP related to ASDs on respiratory and immune systems. Bone marrow-derived antigen-presenting cells (APCs) and splenocytes from atopic prone NC/Nga mice and human airway epithelial cells were exposed to the B.ad or to BaP in the presence and absence of heated-ASDs (H-ASDs). B.ad and BaP in both the presence and absence of H-ASDs increased the expression of cell surface molecules on APCs. H-ASDs alone slightly activated APCs. The expressions induced by B.ad were higher than those induced by BaP in the presence and absence of H-ASDs. There were no remarkable effects on the activation of splenocytes or the proinflammatory responses in airway epithelial cells. These results suggest that B.ad rather than BaP contributes to the exacerbation of asthma regardless of the presence or absence of sand particles, particularly by the activation of the immune system via APCs. Copyright © 2016 John Wiley & Sons, Ltd.

This study examined the relationship between neurobehavioral changes and alterations in gene expression profiles in the brains of mice exposed to different levels of Hg(0) during postnatal development. Neonatal mice were repeatedly exposed to mercury vapor (Hg(0)) at a concentration of 0.057 mg/m(3) (low level), which was close to the current threshold value (TLV), and 0.197 mg/m(3) (high level) for 24 hr until the 20(th) day postpartum. Behavioral responses were evaluated based on changes in locomotor activity in the open field test (OPF), learning ability in the passive avoidance response test (PA), and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. No significant differences were observed in the three behavioral measurements between mice exposed to the low level of Hg(0) and control mice. On the other hand, total locomotive activity in mice exposed to the high level of Hg(0) was significantly decreased and central locomotion was reduced in the OPF task. Mercury concentrations were approximately 0.4 μg/g and 1.9 μg/g in the brains of mice exposed to the low and high levels of Hg(0), respectively. Genomic analysis revealed that the expression of 2 genes was up-regulated and 18 genes was down-regulated in the low-level exposure group, while the expression of 3 genes was up-regulated and 70 genes was down-regulated in the high-level exposure group. Similar alterations in the expression of seven genes, six down-regulated genes and one up-regulated gene, were observed in both groups. The results indicate that an increase in the number of altered genes in the brain may be involved in the emergence of neurobehavioral effects, which may be associated with the concentration of mercury in the brain. Moreover, some of the commonly altered genes following exposure to both concentrations of Hg(0) with and without neurobehavioral effects may be candidates as sensitive biomarker genes for assessing behavioral effects in the early stages of development.

We measured the levels of some biological metals: copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) in the cerebrospinal fluid (CSF) in patients with neurodegenerative diseases (52 patients with amyotrophic lateral sclerosis (ALS)), 21 patients with Alzheimer's disease (AD), and 20 patients with Parkinson's disease (PD) by inductively coupled plasma mass spectrometry (ICP-MS). The diagnoses were additionally supported by neuroimaging techniques for AD and PD. In ALS, the levels of Mg (p

Patients with marked calcification of the basal ganglia and cerebellum have traditionally been referred to as having Fahr's disease, but the nomenclature has been criticized for including heterogeneous etiology. We describe 3 patients with idiopathic bilateral striatopallidodentate calcinosis (IBSPDC). The patients were a 24-year-old man with mental deterioration, a 57-year-old man with parkinsonism and dementia, and a 76-year-old woman with dementia and mild parkinsonism. The former 2 patients showed severe calcification of the basal ganglia and cerebellum, and the latter patient showed severe calcification of the cerebellum. We found significantly increased levels of copper (Cu), zinc (Zn), iron (Fe) and magnesium (Mg), using inductively coupled plasma mass spectrometry in the CSF of all these 3 patients. The increased levels of Cu, Zn, Fe and Mg reflect the involvement of metabolism of several metals and/or metal-binding proteins during the progression of IBSPDC. More numerous patients with IBSPDC should be examined in other races to clarify the common mechanism of the disease and to investigate the specific treatment.

Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn2+, V4+, V5+, Cr3+, Cr6+, Zn2+, Ni2+, and Pb2+ at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V5+ tended to have a greater effect than V4+. The Cr decreased the cell viability, and (Cr+6) at concentrations of 50 and 500 μmol/L was more toxic than (Cr+3). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V+4, V+5), (Cr+3, Cr+6), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals.

Hypoxia is an important modulator of endurance exercise‐induced oxidative adaptations in skeletal muscle. However, whether hypoxia affects resistance exercise‐induced muscle adaptations remains unknown. Here, we determined the effect of resistance exercise training under systemic hypoxia on muscular adaptations known to occur following both resistance and endurance exercise training, including muscle cross‐sectional area (CSA), one‐repetition maximum (1RM), muscular endurance, and makers of mitochondrial biogenesis and angiogenesis, such as peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), citrate synthase (CS) activity, nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), hypoxia‐inducible factor‐1 (HIF‐1), and capillary‐to‐fiber ratio. Sixteen healthy male subjects were randomly assigned to either a normoxic resistance training group (NRT, n =7) or a hypoxic (14.4% oxygen) resistance training group (HRT, n =9) and performed 8 weeks of resistance training. Blood and muscle biopsy samples were obtained before and after training. After training muscle CSA of the femoral region, 1RM for bench‐press and leg‐press, muscular endurance, and skeletal muscle VEGF protein levels significantly increased in both groups. The increase in muscular endurance was significantly higher in the HRT group. Plasma VEGF concentration and skeletal muscle capillary‐to‐fiber ratio were significantly higher in the HRT group than the NRT group following training. Our results suggest that, in addition to increases in muscle size and strength, HRT may also lead to increased muscular endurance and the promotion of angiogenesis in skeletal muscle., This study investigated the effect of resistance exercise training performed under systemic hypoxia or normoxia on biochemical and molecular muscular adaptations in healthy male subjects. Our findings demonstrate that resistance training under systemic hypoxia led not only muscle hypertrophy, but most interestingly, to a greater increase in muscular endurance. This increase in muscular endurance was potentially caused by the increased angiogenesis as determined by capillary‐to‐fiber ratio.

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.

PURPOSE The incidence of blindness is increasing, in part, because of abnormal ocular neovascularization. Anti-VEGF therapies have yielded impressive results; however, they are not a cure for blindness. Recently, metallothioneins (MTs) 1 and 2 have been implicated in the process of angiogenesis. Therefore, we investigated whether MT-1 and MT-2 were also involved in ocular neovascularization. METHODS The concentrations of MT-1 and MT-2 (hereafter MT-1/2) were observed by ELISA. We examined the role of MT-1/2 in ocular neovascularization by using both an oxygen-induced retinopathy (OIR) model and a laser-induced choroidal neovascularization (CNV) model. We investigated the localization of MT-1/2 in retina. Furthermore, we investigated the expression of hypoxia-inducible factor (HIF)-1α and VEGF in OIR. In vitro, we investigated the degradation of HIF-1α. RESULTS The MT-1/2 were significantly elevated in proliferative diabetic retinopathy patients. Ocular neovascularization, which was induced in both the OIR model and the CNV model, was decreased in MT-1/2 knockout (KO) mice. We confirmed that although MT-1/2 was expressed throughout the murine retina, its expression levels were highest in the endothelial cells. Further, OIR enhanced MT-1/2 expression in the retina. Interestingly, in the OIR model, both HIF-1α and VEGF levels were significantly decreased in the retina of MT-1/2 KO mice. In addition, we found that knockdown of MT-1/2 accelerated ubiquitination of HIF-1α. CONCLUSIONS These results indicate that MT-1/2 are involved in retinal and choroidal neovascularization, and that MT-1/2 might be a new therapeutic target in diseases in which ocular angiogenesis is implicated.

A method for the analysis of the sialo-N-glycans in glycoproteins was established by the electrokinetic chromatography mode of capillary electrophoresis (CE) in sodium dodecyl sulfate (SDS) micelles as 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives, using sialo-N-glycans in fetuin as a model. Six major and some minor peaks were observed for the N-glycans in fetuin, which were well separated from each other using 50 mM phosphate buffer, pH 6.0, containing SDS to a concentration of 30 mM in an uncoated fused-silica capillary, and these peaks were assigned to sialo-N-glycans having either of the biantennary or beta1-3/beta1-4 linked galactose-containing complex type triantennary N-glycans as the basic structures, by an indirect method based on the assignment of the peaks in high-performance liquid chromatography separated in parallel with CE and peak collation between these two separation methods. The attaching position of the sialic acid residue was determined using the linkage preference of neuraminidase isozymes. The established system is considered to be useful for routine analysis of microheterogeneity of the carbohydrate moiety of this model glycoprotein from the following reasons: (1) the derivatization with PMP proceeds quantitatively under mild conditions without causing release of the sialic acid residue, (2) the derivatives can be sensitively detected by UV absorption, (3) the procedure is simple, rapid and reproducible. Preliminary results of N-glycan analysis for several other glycoproteins under these conditions are also presented.

In order to elucidate the effect of metallothionein (MT)-III on hepatic gene expression altered by cadmium (Cd), we examined gene expression patterns in the liver of MT-III null mice and wild-type mice after Cd injection using a DNA microarray containing 35,852 genes. In a comparison between Cd-injected MT-III null mice and Cd-injected wild-type mice, 9 genes were found to be up-regulated and 28 genes-including serum amyloid A1 (SAA-1) and SAA-2--were down-regulated.

This study examined the emergence of delayed behavioral effects in offspring mice exposed to low levels of mercury vapor (Hg(0)) during the lactation period. Female offspring of mice were repeatedly exposed to Hg(0) at 0.057 mg/m(3), similar to the current threshold value (TLV), for 24 hr until the 20(th) day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning activity in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at the ages of 3 and 15 months. Hg(0)-exposed mice did not differ from controls in the three behavioral measurements at 3 months of age, and no neurobehavioral effects were observed. On the other hand, the mice exhibited significantly more central locomotion in the OPF task when tested at 15 months of age, but no abnormality in other behavioral performance. Immediately after postnatal exposure, the brain mercury concentration of offspring was about 150 times that of the control, in which the concentrations were approximately 0.4 µg/g. The results indicate that mice exposed to Hg(0) at concentrations around TLV during the developing period resulted in the emergence of delayed behavioral effects at a later stage in life.

DNA microarray containing 30,000 genes was used to monitor the transcriptional response of the neonatal brain after cadmium (Cd) exposure. C57BL/6J pregnant mice were exposed to Cd (10 ppm) during gestation and lactation via drinking water. In a comparison between the Cd-exposed neonatal brain and control, three genes including transferrin receptor (Tfrc) were up-regulated and one gene was down-regulated.

Although cadmium causes hepatotoxicity, its molecular mechanism is unclear. In the present study, transcriptional responses in the liver of C57BL/6J mice given 50 ppm cadmium as a drinking water for 30 days were evaluated with DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not elevated following the administration of cadmium. Cadmium increased the expressions of 2 genes and reduced those of 15 genes in the liver of mice before the leading to hepatotoxicity.

Background & Aims: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis , which might be a useful model of normal ECL cells. Methods: A [ 3 H]PGE 2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP 2 and EP 3 receptor complementary DNAs was conducted. Results: [ 3 H]PGE 2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE 1 = PGE 2 > enprostil >PGF 2α . Although PGE 1 and PGE 2 stimulated 5′-cyclic adenosine monophosphate (cAMP) production, neither PGF 2α nor enprostil had any effect. On the other hand, all of PGE 1 , PGE 2 , PGF 2α , and enprostil attenuated the forskolin-induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE 2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP 2 and EP 3 receptor messenger RNAs was confirmed by RNA blot analysis. Conclusions: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP 2 linked to cAMP production and EP 3 coupled with inhibitory guanosine 5′-triphosphate-binding proteins mediating the inhibition of cAMP production.

To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg(0) and its accumulation as Hg(2+) did not depend on the amount of MT isoform in the tissue, at least in the early phase.

Dental amalgam is a source of exposure to elemental mercury vapor in the general population. The aim of this study was to elucidate the effect of elemental mercury vapor exposure from dental amalgam restorations on gene expression profiles. Out of 26,962 rat genes, mercury vapor was found to increase the expression of 1 gene (Atp1b3) and decrease the expression of 1 gene (Tap1) in the cerebrum, increase the expression of 1 gene (Dnaja2) in the cerebellum, increase the expression of 2 genes (Actb and Timm23) and decrease the expression of 1 gene (Spink3) in the liver, increase the expression of 2 genes (RT1-Bb and Mgat5) and decrease the expression of 6 genes (Tnfaip8, Rara, Slc2a4, Wdr12, Pias4 and Timm13) in the kidney.

Cadmium (Cd) is a toxic heavy metal that has been shown to induce vascular diseases, such as atherosclerosis. We used DNA microarray to monitor the transcriptional response of human coronary artery endothelial cells (HCAEC) to a non-lethal dose of Cd (10 µM). Out of 35,035 human genes, Cd enhanced the expression of 3 metallothionein (MT)-I subisoform genes, including MT1E, MT1H and MT1B, and reduced the expression of 12 genes, including ISG20 and TK1, 2-fold or greater.

Aims In order to evaluate the role of metallothionein (MT)-III in cadmium (Cd)-induced testicular toxicity, we examined the sensitivity of MT-III null mice to severe testicular injury caused by Cd. Main methods Male MT-III null mice, MT-I/II null mice and wild-type mice were given a subcutaneous injection of CdCl2 (15 μmol/kg). The testis was collected from each mouse at 6, 12 and 24 h after Cd administration. Key findings Testicular hemorrhages by evaluating morphology, hemoglobin content and histological parameters in the 3 types of mice were elevated by Cd injection in a time-dependent manner. The degree of hemorrhage in Cd-injected MT-I/II null mice was similar to that in the wild-type mice. In contrast, hemorrhage in the MT-III null mice was attenuated compared with that in wild-type mice and MT-I/II null mice. Cd levels, MT-I and MT-II mRNA levels and Cd-binding molecules in the testis were similar between MT-III null mice and wild-type mice. In microarray analysis, high expression of purine-nucleoside phosphorylase 2 (Pnp2), retinal degeneration 3 (Rd3), and cadherin-like 24 (Cdh24) was revealed in the testis of MT-III null mice under normal or Cd-treated conditions. Significance MT-III null mice were found to show attenuation of Cd-induced severe testicular toxicity. These results suggest the lack of MT-III contributes to protection of testis from Cd. In addition, regulation of Pnp2, Rd3, and Cdh24 mRNA levels may involve the sensitivity of MT-III null mice to Cd.

DNA microarray was used to monitor the transcriptional response of human brain microvascular endothelial cells (HBMEC) and human coronary artery endothelial cells (HCAEC) to sodium arsenite (iAs(III)). iAs(III) enhanced the expression of 10 and 6 genes, and reduced the expression of 45 and 20 genes in HBMEC and HCAEC, respectively. Among the 64 genes whose expression was changed in some way, HBMEC and HCAEC had 5 up-regulated and 12 down-regulated genes in common.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A λgt11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity ( T. Nomura, T. Fukui, and A. Ichikawa, 1991 , Biochim. Biophys. Acta 1077, 47–55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5′ and 3′ noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus , and Dictyostelium discoideum NDP kinases, as well as the human Nm 23-gene product and the awd protein of Drosophila melanogaster .

A cDNA clone for the mouse thromboxane A2 receptor was isolated from mouse lung cDNA library. The cDNA has a 1,023 base pair open reading frame which encodes a protein of 341 amino acid residues. STA2 and U-46619 induced inward current in Xenopus laevis oocytes injected with the transcript of the clone. Specific binding of [3H]S-145 was found in membranes of COS-1 cells transfected with the cDNA (Kd = 3.3 nM) and was displaced with unlabeled prostaglandins and thromboxane analogues in the order of S-145 greater than STA2 greater than U-46619 greater than PGD2 greater than PGF2 alpha = PGE2. Northern blot analysis demonstrated that thromboxane A2 receptor mRNA is expressed abundantly in thymus, spleen and lung.

Urinary excretion of human liver-type fatty acid-binding protein (hL-FABP), which is expressed in human proximal tubules and engaged in free fatty acid (FFA) metabolism, was reported to reflect the clinical prognosis of chronic kidney disease. Here we have investigated the pathophysiological significance of hL-FABP in a model of protein overload nephropathy. Because L-FABP is not expressed in the wild-type mice, we generated hL-FABP chromosomal gene transgenic (Tg) mice. Tg mice were intraperitoneally injected with bovine serum albumin (BSA) replete with FFAs (r-BSA group) or FFA-depleted BSA (d-BSA group). The r-BSA group developed significantly more severe tubulointerstitial damage than did the d-BSA group. Renal expression of the hL-FABP gene was more up-regulated, and urinary excretion of hL-FABP was significantly higher, in the r-BSA group than in the d-BSA group. Furthermore, compared with their wild-type littermates injected with r-BSA, the number of infiltrated macrophages was significantly attenuated in Tg mice injected with it on day 28. In patients with kidney disease (n = 50), urinary hL-FABP was correlated with both urinary protein and the severity of tubulointerstitial injury. In conclusion, our experimental model suggests that urinary excretion of hL-FABP reflects stresses, such as urinary protein overload, on the proximal tubules. The clinical observations support this hypothesis.

The purpose of this study was to examine the role of muscarinic cholinergic and alpha2-adrenergic mechanisms in growth hormone (GH) secretion during exercise in humans. The GH responses induced during moderate-intensity exercise (using a cycle ergometer at 60% maximal oxygen uptake, VO2max, for 30 min) without treatment (control) and after the administration of a muscarinic cholinergic antagonist (atropine 1 mg) or after an alpha2-adrenergic antagonist (yohimbine 15 mg) were compared in seven healthy men. Although, serum GH concentration had increased significantly after exercise in the control experiment [mean peak GH concentration 52.64 (SEM 18.60) ng x ml(-1)], the increase was suppressed by the administration of either atropine [mean peak GH concentration 8.64 (SEM 7.47) ng x ml(-1)] or yohimbine [mean peak GH concentration 17.50 (SEM 7.89) ng x ml(-1)]. The area under the curve of serum GH concentration against time was significantly lower in the experiment using these drugs [with atropine, mean area 458 (SEM 409) ng x ml(-1) min], with yohimbine mean area 946 (SEM 435) ng x ml(-1) min] than in the control experiment [mean area 3135 (SEM 1098) ng x ml(-1) x min]. These results suggest that muscarinic cholinergic and alpha2-adrenergic mechanisms are involved in GH secretion during exercise in humans.

PURPOSE. Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteinerich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage. METHODS. The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage. RESULTS. The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III‐ deficient mice was remarkably thinner compared to lightexposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II‐deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure. CONCLUSIONS. These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/ II. Some of these effects may be exerted by its antioxidative potency. (Invest Ophthalmol Vis Sci. 2012;53:7896‐7903)

Thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) are two of the most representative eicosanoids that are formed from arachidonic acid. They produce a broad spectrum of biological effects mediated through specific cell surface receptors. We have mapped genetic loci for TXA2 receptor, Tbxa2r, and for PGE2 receptor subtypes EP2 and EP3, Ptgerep2 and Ptgerep3, respectively, using restriction fragment length variants in interspecific backcross mice. None of the three loci cosegregated with each other. Tbxa2r mapped to Chr 10, Ptgerep2 mapped to the centromeric region of Chr 15, and Ptgerep3 mapped to the distal end of Chr 3. Possible human loci for these receptors are predicted based on the homology between mouse and human chromosomes.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

The functional interaction of prostaglandin E (PGE) receptor EP 3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP 3 receptor cDNA-transfected Chinese hamster ovary cells. PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP 3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE 2 binding to the membranes was increased by GTPγS, and PT treatment also increased the binding activity to the same level as that increased by GTPγS, but the sensitivity of GTPγS was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE 2 binding activity with the following preference: Gi 1 = Gi 2 > Gi 3 > Go , but GTPγS completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in K d without the change of B max , and due to suppression of association rate. [ 3 H]PGE 2 -bound EP 3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTPγS was eluted at the position of M r = approx . 60 kDa, similar to the relative molecular mass of EP 3 receptor deduced from its amino acid sequence. In contrast, [ 3 H]PGE 2 -bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M r = approx . 130 kDa, corresponding to the M r of the complex of EP 3 receptor and Gi2, but GTPγS shifted the position of its elution from M r = 130 to 60 kDa. Furthermore, addition of PGE 2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi 1 = Gi 2 > Gi 3 > Go . These results indicate that EP 3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.

The component aldoses, uronic acid, and hexosamines in the total non-dialyzable urinary glycoconjugates were determined by the dithioacetal method, and normal levels of these monosaccharides are presented. The molar ratios of fucose/galactose, glucuronic acid/galactose, and galactosamine/glucosamine for cancer patients were lower, and that of mannose/galactose was higher, than normal values.

We present herein transcriptional changes in mouse spinal cords in response to physical exercise on a treadmill using a DNA microarray. By 30-min exercise, the expression of 3 genes was enhanced and expression of 29 genes reduced. By continuous 2-week exercise (30-min exercise per day), the expression of 1 gene was enhanced and expression of 13 genes reduced.