池上 昭彦

Focusing on the relatively unexplored presence of micro- and nano-plastic aerosol particles, this study quantitatively assessed the emission of nano-plastic particles during the machining of carbon fiber reinforced plastic (CFRP) in the working environment. Measurements of aerosol particles smaller than 1 µm in size were performed by aerosol mass spectrometry. The findings revealed that concentrations of carbonous aerosol particles (organic aerosol and refractory black carbon (rBC)) were higher during working hours than during non-working hours. Positive matrix factorization identified CFRP particles as a significant source, contributing an average of approximately 30% of concentration of carbonous aerosol particles during working hours. This source apportionment was corroborated by the presence of bisphenol A and F fragments, principal components of the epoxy resins used in CFRP, and was corroborated by similarities to the carbon cluster ion distribution observed in rBC during CFRP pipe-cutting operations. Further, the particle size distribution suggested the existence of plastic aerosol particles smaller than 100 nm. This study established the method to quantitatively distinguish nano-plastic aerosol particles from other aerosol particles in high temporal resolution and these techniques are useful for accurately assessing exposure to nano-plastic aerosol particles in working environments.

OBJECTIVES: Carbon fibers are used in a variety of industrial applications, based on their lightweight and high stiffness properties. There is little information on the characteristics and exposure levels of debris generated during the factory processing of carbon fibers or their composites. This study revisits the general assumption that carbon fibers or their debris released during composite processing are considered safe for human health. METHODS: The present interventional study was conducted at a factory located in Japan, and involved on-site collection of debris generated during the industrial processing of polyacrylonitrile (PAN)-based carbon-fiber-reinforced plastic (CFRP). The debris were collected before being exhausted locally from around different factory machines and examined morphologically and quantitatively by scanning electron microscopy. The levels of exposure to respirable carbon fibers at different areas of the factory were also quantified. RESULTS: The collected debris mainly contained the original carbon fibers broken transversely at the fiber's major axis. However, carbon fiber fragments morphologically compatible with the WHO definition of respirable fibers (length: > 5 μm, width: < 3 μm, length/width ratio: > 3:1) were also found. The concentrations of respirable fibers at the six examined factory areas under standard working conditions in the same factory were below the standard limit of 10 fibers/L, specified for asbestos dust-generating facilities under the Air Pollution Control Law in Japan. CONCLUSIONS: Our study identified potentially dangerous respirable fibers with high aspect ratio, which was generated during the processing of PAN-based CFRP. Regular risk assessment of carbon fiber debris is necessary to ensure work environment safety.

BACKGROUND: Allergen-specific immunoglobulins have a crucial role in allergic diseases. Most wheeze episodes develop before school age, and allergic rhinitis later develops during early elementary school years. However, the clinical background and cytokine/chemokine profiles associated with changes in immunoglobulins during early school-age are poorly understood. METHODS: This study used blood samples from children participating in the JECS Pilot Study. We examined nineteen kinds of aeroallergen-specific immunoglobulins (IgE, IgG1, IgG4, and IgA) levels in patients at age 6 and age 8. Fluctuations of Der f 1- and Cry j 1-specific immunoglobulins levels during the two periods were compared to assess the frequency of allergic statuses and clusters of cytokine/chemokine profiles. RESULTS: The medians of aeroallergen-specific IgE levels did not fluctuate, and almost all IgG1 and IgG4 decreased. In IgA, four (e.g., Der f 1) increased, whereas the other four (e.g., Cry j 1) decreased. The ratio of the Der f 1-specific IgG1 level at age 8 to that at age 6 was higher in children with poor asthma control than in children with better asthma control. Moreover, the cytokine/chemokine cluster with relatively lower IL-33 and higher CXCL7/NAP2 was associated with lower Der f 1- and Cry j 1-specific IgG4 levels, but not IgE levels. CONCLUSIONS: The cluster of cytokine/chemokine profiles characterized by lower IL-33 and higher CXCL7/NAP2 was associated with the maintenance of aeroallergen-specific IgG4 levels. This result provides a basis for considering the control of aeroallergen-specific immunoglobulins.

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 μM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.

Objective: Ultra-sensitive hormone assays have detected slight sex differences in blood estradiol (E2) levels in young children before adrenarche. However, the origin of circulating E2 in these individuals remains unknown. This study aimed to clarify how E2 is produced in young girls before adrenarche. Design: This is a satellite project of the Japan Environment and Children's Study organized by the National Institute for Environmental Studies. Methods: We collected blood samples from healthy 6-year-old Japanese children (79 boys and 71 girls). Hormone measurements and data analysis were performed in the National Institute for Environmental Studies and the Medical Support Center of the Japan Environment and Children's Study, respectively. Results: E2 and follicle stimulating hormone (FSH) levels were significantly higher in girls than in boys, while dehydroepiandrosterone sulfate (DHEA-S) and testosterone levels were comparable between the two groups. Girls showed significantly higher E2/testosterone ratios than boys. In children of both sexes, a correlation was observed between E2 and testosterone levels and between testosterone and DHEA-S levels. Moreover, E2 levels were correlated with FSH levels only in girls. Conclusions: The results indicate that in 6-year-old girls, circulating E2 is produced primarily in the ovary from adrenal steroids through FSH-induced aromatase upregulation. This study provides evidence that female-dominant E2 production starts several months or years before adrenarche. The biological significance of E2 biosynthesis in these young children needs to be clarified in future studies.

Prenatal exposure to methylmercury (MeHg) affects child development after birth. However, many epidemiological studies have evaluated total mercury levels without analyzing speciation. Biomonitoring of MeHg and inorganic mercury (IHg) is essential to reveal each exposure level. In this study, we compared a high-throughput analysis for mercury speciation in blood using liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) and liquid chromatography-cold vapor atomic fluorescence spectrometry (LC-CVAFS). The validated LC-ICP-MS method was applied to 101 maternal blood and 366 cord blood samples in the pilot study of the Japan Environment and Children's Study (JECS). The accuracy of the LC-CVAFS method ranged 90-115% determined by reference material analysis. To evaluate the reliability of 366 cord blood samples, fifty cord blood samples were randomly selected and analyzed using LC-CVAFS. The median (5th-95th percentile) concentrations of MeHg and IHg were 5.4 (1.9-15) and 0.33 (0.12-0.86) ng/mL, respectively, in maternal blood, and 6.3 (2.5-15) and 0.21 (0.08-0.49) ng/mL, respectively, in cord blood. Inter-laboratory comparison showed a relatively good agreement between LC-ICP-MS and LC-CVAFS. The median cord blood:maternal blood ratios of MeHg and IHg were 1.3 and 0.5, respectively. By analyzing speciation, we could focus on the health effects of each chemical form.

Fusobacterium nucleatum is a human periodontal pathogen that causes opportunistic infections. It has been implicated in preterm birth and has as a pathogen of colorectal cancer. However, it is a common member of the oral microbiota and can have a symbiotic relationship with its hosts. To date, studies of F. nucleatum have been hindered by a lack of effective genetic tools, and the transformation of F. nucleatum has not been investigated. In this chapter, protocols for the transformation of F. nucleatum strain 12230 using sonoporation are presented. We also include a genetic complementation protocol for a F. nucleatum knockout mutant.

BACKGROUND: The relationship between allergic individuals and their responsiveness to routine vaccines has rarely been investigated. This study examined whether the seroprevalence of measles antibody differed between children with and without allergic diseases in the general pediatric population. METHODS: The cross-sectional study was performed within a prospective general birth cohort (a pilot study of the Japan Environment & Children's Pilot Study [JECS]) of children aged 8 years. The clinical history of allergic diseases, measles, and the concentration of measles immunoglobulin G titers in serum enzyme immunoassay were examined. Fisher's exact tests were used to assess the relationships between the allergic characteristics of the children and their measles antibody positivity rates. RESULTS: This study included 162 children. Any allergic disease was reported in 75 (46.3%). The measles antibody positivity rate was 94.7% among children with any allergic diseases and 92.0% among children without allergic diseases. Our results revealed no differences in measles antibody seropositivity between children with allergies and controls. CONCLUSIONS: Children with allergies mount and maintain a comparable immune response to the measles vaccine.

Nano-sized titanium dioxide (TiO2) particles are produced on a large scale and are widely used. However, the effects of exposure to nano-sized TiO2 particles on the cardiovascular system remain elusive. The present study investigated how the crystal structure and surface characteristics of nano-sized TiO2 particles affect the adhesion of monocytes to endothelial cells, which is an essential process in atherosclerogenesis. Human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1) were exposed to anatase (NM102), rutile hydrophobic (NM103), and rutile hydrophilic (NM104) TiO2 nanoparticles. Incubation of HUVECs with NM102, NM103, or NM104 at 100 mu g/ml significantly reduced cell viability, and cell viability was also significantly reduced after exposure to 75 mu g/ml of the anatase particles (NM102). Brief exposure to NM102 and NM103 increased the level of intercellular adhesion molecule-1 (ICAM-1) in HUVECs. Exposure to NM102 also upregulated the expression of lymphocyte function-associated antigen-1 (LFA-1) in THP-1 cells, resulting in enhanced adhesion of these cells to HUVECs. The amount of titanium uptake of NM102 in cells was higher than that of NM103 or NM104, as evidenced by inductively coupled plasma optical emission spectroscopy (ICP-OES). To examine the effects of long-term exposure to nano-sized TiO2 on the process of atherosclerosis, apolipoprotein E deficient (ApoE(-/-)) mice were exposed to TiO2 NPs (NM102, NM103, or NM104 at 10 or 40 mu g/mouse) by pharyngeal aspiration once every other week for 10 weeks. As the results, NM102 (40 mu g/mouse) increased plaque formation in the aorta, and this increase correlated with a significant upregulation of ICAM-1 and F4/80 expression in the aorta. Anatase, but not rutile, TiO2 nanoparticles promoted the adhesion of monocytes to endothelial cells, and enhanced atherosclerogenesis in a susceptible animal model.

Lead exposure is associated with impaired neurodevelopment among children. House dust is recognized as one of the important secondary sources of lead exposure in children. We assessed the relationship between lead contamination in house dust and blood lead level in Pakistani children. We investigated lead contamination in house dust samples collected from 59 houses in Karachi, Pakistan. The lead content of house dust in Pakistan was relatively higher than that reported in previous studies. Weekly lead intakes from house dust were considerably higher among Pakistani children. In Pakistani children, 12% (7 of 58) showed lead intake values greater than the previous Provisional Tolerable Weekly Intake of lead. A correlation (Pearson's correlation = 0.37) was found between weekly lead intake from house dust and blood lead level in Pakistani children. In addition, blood lead levels were significantly higher in children with high lead intakes than in children with low and medium lead intakes. Thus, house dust is an important source of lead exposure in Pakistani children.

Objectives Exposure to inorganic arsenic (iAs) is a world-wide health concern. We reported that Japanese children and pregnant women are exposed to moderate levels of iAs through food. Reducing iAs contamination from foods of high iAs is an important issue unique in Japan. Integrated iAs is methylated to less toxic organic forms, and S-adenosyl-L-methyonine (SAM), a common methyl-donor of DNA and histones, is utilized in this process. Chronic consumption of SAM by iAs metabolism due to exposure to iAs might alter the epigenetic modification of genome. The SAM biosynthesis pathway is dependent on folate cycle, and it is possible that ingestion of sufficient folic acid (FA) is protective to iAs induced toxicity. Methods In the course of our cross-sectional body burden analyses of Pb and iAs in Japanese children and pregnant women, termed "PbAs study", FA concentration in serum of 104 pregnant women was measured. Results Mean (±SEM) of serum FA concentration was 15.8 ± 1.3 (ng/mL). There are significant number of people showing very high FA (>30 ng/ mL), and large fraction of them were taking supplements daily. Conclusions These results suggested that level of FA ingestion of Japanese pregnant women is high for supporting normal fetal development.

Nanoparticles (NPs) are widely used in food, and analysis of their potential gastrointestinal toxicity is necessary. The present study was designed to determine the effects of silica dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) NPs on cultured THP-1 monocyte-derived macrophages and human epithelial colorectal adenocarcinoma (Caco-2) cells. Exposure to ZnO NPs for 24 h increased the production of redox response species (ROS) and reduced cell viability in a dose-dependent manner in THP-1 macrophages and Caco-2 cells. Although TiO2 and SiO2 NPs induced oxidative stress, they showed no apparent cytotoxicity against both cell types. The effects of functionalized SiO2 NPs on undifferentiated and differentiated Caco-2 cells were investigated using fluorescently labeled SiO2 NPs with neutral, positive, or negative surface charge. Exposure of both types of cells to the three kinds of SiO2 NPs significantly increased their interaction in a dose-dependent manner. The largest interaction with both types of cells was noted with exposure to more negatively surface-charged SiO2 NPs. Exposure to either positively or negatively, but not neutrally, surface-charged SiO2 NPs increased NO levels in differentiated Caco-2 cells. Exposure of differentiated Caco-2 cells to positively or negatively surface-charged SiO2 NPs also upregulated interleukin-8 expression. We conclude that functionalized surface-charged SiO2 NPs can induce pro-inflammatory responses but are noncytotoxic.

This study aimed to reveal a new dimension of allergy profiles in the general population by using machine learning to explore complex relationships among various cytokines/chemokines and allergic diseases (asthma and atopic dermatitis; AD). We examined the symptoms related to asthma and AD and the plasma levels of 72 cytokines/chemokines obtained from a general population of 161 children at 6 years of age who participated in a pilot birth cohort study of the Japan Environment and Children's Study (JECS). The children whose signs and symptoms fulfilled the criteria of AD, which are mostly based on questionnaire including past symptoms, tended to have higher levels of the two chemokine ligands, CCL17 and CCL27, which are used for diagnosis of AD. On the other hand, another AD-related chemokine CCL22 level in plasma was higher only in children with visible flexural eczema, which is one of AD diagnostic criteria but was judged on the same day of blood examination unlike other criteria. Here, we also developed an innovative method of machine learning for elucidating the complex cytokine/chemokine milieu related to symptoms of allergic diseases by using clustering analysis based on the random forest dissimilarity measure that relies on artificial intelligence (AI) technique. To our surprise, the majority of children showing at least any asthma-related symptoms during the last month were divided by AI into the two clusters, either cluster-2 having elevated levels of IL-33 (related to eosinophil activation) or cluster-3 having elevated levels of CXCL7/NAP2 (related to neutrophil activation), among the total three clusters. Future studies will clarify better approach for allergic diseases by endotype classification.

Poly- and perfluoroalkyl substances (PFAS) have been investigated in a number of cohort studies due to concern over their adverse health effects. The aim of this study was to develop a reliable, high throughput and cost-effective analytical method for a broad range of PFAS in human serum. Protein precipitation, automatic solid phase extraction (SPE) pre-treatment and column-switching LC-MS/MS were employed. The optimised and validated method was then used to analyse the levels of 28 PFAS in 339 maternal serum samples from Pilot Study of the Japan Environment and Children's Study. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnA) and perfluorooctane sulphonic acid (PFOS) were detected in all 339 samples at median (range) concentrations of 1.9 (0.46-15), 1.5 (0.32-10), 1.3 (0.25-4.5) and 3.7 (0.43-15) ng/ml, respectively. These levels are comparable to those reported in previous studies using samples collected from various parts of the world. With a few exceptions, the remainder of the PFAS examined had lower detection rates but were found at concentrations similar to those reported in previous studies. The sensitivity and throughput ability of the method developed here are sufficient for its application in a large-scale biomonitoring study.

OBJECTIVES: No previous study has used repeated measures data to examine the associations of dog/cat ownership with wheezing and asthma prevalence among children. This prospective study used repeated measurers analysis to determine whether dog/cat ownership in childhood is an independent risk factor for wheezing and asthma, after adjustment for gestational, socio-economical, and demographical confounders confounders, in Japan. METHODS: We conducted a multicenter pilot study of the Japan Environment and Children's Study (JECS) during 2009-2010. Among 440 newborn infants enrolled, 410 (52.8% males) were evaluated for dog/cat ownership in the home and history of wheezing and asthma in five follow-up questionnaire surveys (until age 6 years). Dog/cat ownership during follow-up period was categorized into four groups: 7.6% were long-term dog/cat owners, 5.9% were toddler-age owners, 5.9% were preschool-age owners, and 80.7% were never owners. RESULTS: The prevalence of wheezing during follow-up period increased from 20.8% to 35.4% and the prevalence of asthma increased from 1.3% to 16.3%. A fitted logistic generalized estimating equation models including important confounders showed no significant associations of the interaction between dog and/or cat ownership and follow-up time with the risks of wheezing and asthma. However, the risks of wheezing and asthma were slightly lower for long-term and toddler-age dog/cat owners than for preschool-age and never owners. CONCLUSIONS: The present findings suggest that dog and cat ownership from toddler-age does not increase the risks of wheezing and asthma compared with never owners among Japanese children.

Lead (Pb) in petrol has been banned in developed countries. Despite the control of Pb in petrol since 2001, high levels were reported in the blood of pregnant women and children in Pakistan. However, the identification of sources of Pb has been elusive due to its pervasiveness. In this study, we assessed the lead intake of pregnant women and one- to three-year-old children from food, water, house dust, respirable dust, and soil. In addition, we completed the fingerprinting of the Pb isotopic ratios (LIR) of petrol and secondary sources (food, house-dust, respirable dust, soil, surma (eye cosmetics)) of exposure within the blood of pregnant women, newborns, and children. Eight families, with high (~50 µg/dL), medium (~20 µg/dL), and low blood levels (~10 µg/dL), were selected from 60 families. The main sources of exposure to lead for children were food and house-dust, and those for pregnant women were soil, respirable dust, and food. LIR was determined by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) with a two sigma uncertainty of ±0.03%. The LIR of mothers and newborns was similar. In contrast, surma, and to a larger extent petrol, exhibited a negligible contribution to both the child’s and mother’s blood Pb. Household wet-mopping could be effective in reducing Pb exposure. This intake assessment could be replicated for other developing countries to identify sources of lead and the burden of lead exposure in the population.

Adverse health effects of heavy metals are a public health concern, especially lead may cause negative health impacts to human fetal and infantile development. The lead concentrations in Pakistani pregnant women's nails, used as a biomarker, were measured to estimate the lead exposure. Thirteen nail samples out of 84 nails analyzed contained lead higher than the concentration (13.6 mu g/g) of the fatal lead poisoning case, raising the possibility of an external contamination. Eye cosmetics such as surma are recognized as one of the important sources of lead exposure in Pakistan. We collected in Pakistan 30 eye cosmetics made in Pakistan, Saudi Arabia and western countries. As the metal composition analysis by energy dispersive X-ray fluorescence spectrometry revealed that some surma samples contained lead more than 96%, the surma might contaminate the nail specimen. Scanning electron microscopy observations showed that lead-containing surma consists of fine particle of galena (ore of lead sulfide) in respirable dust range (less than 10 mu m). In addition, relative in vitro bioavailability of lead in the surma was determined as 5.2%. Thus, lead-containing surma consists of inhalable and bioavailable particles, and it contributes an increased risk of lead exposure. Moreover, the relationship between the surma and the lead-contaminated nails by lead isotope ratios analysis indicated the potential of lead contamination in nails by surma. These results suggest that lead in the nails was derived both from body burden of lead and external contamination by lead-containing surma. Therefore, nail is not suited as a biomarker for lead exposure in the countries where surma used, because we may overestimate lead exposure by surface lead contamination in the nail by surma. (C) 2016 Elsevier Ltd. All rights reserved.

This study aimed to evaluate the relationships between oxidative stress and heavy metal exposure (lead [Pb] and cadmium [Cd]), as well as co-factors such as physical activity and age, in Japanese women. This study was conducted with female subjects from a rural agricultural community in Japan. Subjects were asked to complete lifestyle-related questionnaires and undergo a group health examination. Physical activity, alcohol consumption, body mass index, and other demographic information were collected. Blood and urine samples were collected to measure urinary 8-hydroxydeoxyguanosine (8-OHdG) levels and blood and urinary Cd and Pb concentrations. Urine samples were analyzed using high performance liquid chromatography and flameless atomic absorption spectrometry; blood samples were analyzed using inductively coupled plasma-mass spectrometry. Age, physical activity, and blood and urinary Cd and Pb concentrations were included in structural equation modeling analysis. Two latent factors for heavy metal exposure and physical activity were produced to predict the total influence of the variables. The final model was good: CMIN/DF = 0.775, CFI = 1.000, GFI = 0.975, AGFI = 0.954, RMSEA = 0.000. 8-OHdG levels were positively associated with heavy metal exposure, physical activity, and age (standard beta of path analysis: 0.33, 0.38, and 0.20, respectively). Therefore, oxidative stress is associated with both, environmental and lifestyle factors, in combination with aging.

Aim: Exposure assessment of lead (Pb) and Arsenic (As) from food, water, and house dust intake were assessed among pregnant women, their children and fetuses in Pakistan and Japan, as well as their body burden of the metals in their blood. Method: Fifty families which included a pregnant woman, a fetus and the 1-3-year-old siblings were recruited in Karachi and Khairpur in Pakistan, and Shimotsuke and Asahikawa in Japan, respectively. Their dietary exposure to Pb and As was measured in 3-day food duplicates and drinking water by ICP-MP. Pb in house dust and respirable dust was evaluated with an energy dispersive X-ray fluorescence spectrometry. Nonradioactive isotope Pb profiles of blood specimens will be compared with those of the exposure origins, such as food duplicates, respirable house dust, the soils nearby, and gasoline. Results: Judging from the data collected and analyzed so far, contribution from dietary intake is highly correlated to higher body burden of Pb among Pakistani mothers. Additional data analyses will reveal the status of Pb and As body burden in Pakistani mothers, fetuses and their siblings, and causal sources of high body burden is delineated by Pb isotope profile analysis of different sources of Pb exposure.

To measure current Hg, Cd, and Pb exposure in Japanese children, and to estimate dietary intakes of foods responsible for high body burden. Blood, hair, and urine samples were collected from 9 to 10-year-old 229 children in Asahikawa and measured for Hg, Cd, and Pb in these matrices. Diet history questionnaire was used to estimate intake of marine foods and other food items. Hg level was measured by cold vapor atomic absorption spectrometry. Cd and Pb levels were determined with inductively coupled plasma mass spectrometry. Geometric mean (GM) of blood Hg, Cd, and Pb was 4.55 mu g/L, 0.34 mu g/L, and 0.96 mu g/dL, respectively. Urinary Cd level was 0.34 mu g/g creatinine (GM) and hair Hg was 1.31 mu g/g (GM). Approximately one-third (35 %) of blood samples had Hg level above the U.S. EPA reference dose (RfD; 5.8 mu g/L). Hair Hg level exceeded U.S. EPA RfD (1.2 mu g/g) in 59 % samples. Children in the upper quartile of blood Hg level had significantly higher intake of large predatory fish species compared to those in the lower quartile of blood Hg. Those with high blood Hg level may be explained by more frequent intake of big predatory fish. Cd and Pb exposure is generally low among Japanese children. As no safety margin exists for Pb exposure and high exposure to MeHg is noted in Japanese population; periodic biomonitoring and potential health risk assessment should continue in high-risk populations, notably among children.

Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 angstrom crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel alpha-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the "leucine chain." The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.

Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.

intrauterine infection is a recognized cause of preterm birth. The infectious organisms are believed to originate primarily from the vaginal tract and secondarily from other parts of the body. It is plausible that microbes in the oral cavity can be transmitted to the pregnant uterus. However, direct evidence supporting such a transmission is lacking. In this study, amniotic fluids of 34 pregnant women were examined by PCR using 16S and 23S rRNA universally conserved primers. Bacterial DNA was amplified from the only patient with clinical intrauterine infection and histologic necrotizing acute and chronic chorioamnionitis. One strain, Bergeyella sp. clone AF14, was detected and was 99.7% identical to a previously reported uncultivated oral Bergeyella strain, clone AK152, at the 16S rRNA level. The same strain was detected in the subgingival plaque of the patient but not in her vaginal tract. The 16S-23S rRNA sequence of clone AF14 matched exactly with the sequences amplified from the patient's subgingival plaque. These observations suggest that the Bergeyella strain identified in the patient's intrauterine infection originated from the oral cavity. This is the first direct evidence of oral-utero microbial transmission. The patient's periodontal health during pregnancy was unclear. She did not have detectable periodontal disease during postpartum examination. Bergeyella spp. had not been previously associated with preterm birth and were detected in subgingival plaque of women without clinical levels of intrauterine infection. Uncultivated species may be overlooked opportunistic pathogens in preterm birth. This study sheds new light on the implication of oral bacteria in preterm birth.

Background: Periodontitis develops as a result of the interaction of the host with subgingival plaque bacteria. Both Porphyromonas gingivalis and Treponema denticola are frequently associated together in these oral biofilms. Methods: The molecular basis for in vitro biofilm formation was investigated for P. gingivalis 38 1, T. denticola. 35405, and mixtures of the two organisms using microtiter plate assays. In addition, the biofilms were examined following confocal laser scanning microscopy. Results: P. gingivalis 381, but not T. denticola strains, formed biofilms in vitro. This property was dependent, in part, on the strain 381 fimA, ppk, and usp genes. Microarray and Northern blot analyses suggested that the expression of the ppk gene was required for maximal expression of the uspA gene. P. gingivalis 381 formed synergistic biofilms when incubated with T. denticola strains. This process was dependent upon the strain 381 rgpB and fimA genes as well as the T. denticola flgE and cfpA genes. Conclusions: P. gingivalis 381 formed synergistic biofilms with T. denticola 35405. These results may be relevant to the previous observations that the two organisms are frequently observed together in subgingival plaque with the spirochetes localized to the exterior of the oral biofilms. It is suggested that other such synergistic effects may also occur between other plaque bacteria.

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T denticola to form a mixed biofilm. The T denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium navi-forme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

Deep-sea bacteria have unique systems for gene and protein expression controlled by hydrostatic pressure. One of the sigma factors, sigma(54), was found to play an important role in pressure-regulated transcription in a deep-sea piezophilic bacterium, Shewanella violacea. A glutamine synthetase gene (glnA) has been targeted as a model for the pressure-regulated promoter to investigate transcriptional regulation by the sigma(54) factor. Recognition sites for sigma(54) and sigma(70) factors were observed at an upstream region of the glnA, and NtrC-binding sites were also identified at the same region. Primer extension analyses revealed that the transcription initiation sites of both promoters were determined and that transcription from the sigma(54) site was regulated by elevated pressure. The sigma(54) promoter is known to be activated by a two-component signal transduction system, the NtrB-NtrC phosphorylation relay. Our results suggested that this system might be regulated by deep-sea conditions and that the gene expression controlled by the sigma(54) promoter was actually regulated by pressure. We propose a possible model of the molecular mechanisms for pressure-regulated transcription.

Deep-sea bacteria have unique systems for gene and protein expression controlled by hydrostatic pressure. One of the sigma factors, sigma(54), was found to play an important role on the pressure-regulated transcription in a deep-sea piezophilic bacterium, Shewanella violacea. A glutamine synthetase gene (glnA) has been targeted as a model for the pressure-regulated promoter to investigate the transcriptional regulation by the sigma(54) factor. Recognition sites for sigma(54) and sigma(70) factors were observed at an upstream region of the glnA and also NtrC-binding sites were identified at this same region. Primer extension analyses revealed that the transcription initiation sites of both promoters were determined and that the transcription from the sigma(54) site was regulated by elevated pressure. The sigma(54) promoter is known to be activated by a two components signal transduction system, NtrB-NtrC phospholylated relay. Our results suggested that this system might be regulated by deep-sea conditions and that the gene expression controlled by the sigma(54) promoter was actually regulated by pressure. We proposed a possible model of the molecular mechanisms for pressure-regulated transcription.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively, Comparison with E, coli sigma (70) and P. putida sigma (70) showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S, violacea sigma (70) factor was overexpressed in E, coli and successfully purified to near homogeneity.

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-sea piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino acids with a molecular mass of 36 238 Da. S. violacea alpha protein was expressed in a ts Escherichia coli mutant. to confirm whether the rpoA gene is functional. It complemented this mutation, indicating a chimeric RNA polymerase is assembled at the non-permissive temperature. Recombinant alpha protein was overexpressed using an expression plasmid harboring the rpoA gene and purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma (70) RNA polymerase. It also indicated that pressure response (piezoresponse) in the alpha operon occurred at the transcriptional level. suggesting some positive regulators may interact with the transcriptional apparatus and regulate the expression of the operon at different pressure conditions. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

A glutamine synthetase gene (glnA) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb SacI fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli. Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by sigma (70) and sigma (54) were identified in the region upstream of glnA. An electrophoretic mobility shift assay demonstrated that S. violacea sigma (54) specifically binds to the promoter region of glnA, suggesting that sigma (54) may play an important role in pressure-regulated transcription in this piezophilic bacterium. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55 359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon. (C) 2000 Elsevier Science B.V. All rights reserved.

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.