Predictive Evaluation of Pharmaceutical Properties of Ulinastatin-Containing Vaginal Suppositories as a Hospital Preparation by Near-Infrared Spectroscopy.

A vaginal suppository containing ulinastatin (UTI) was developed as a hospital pharmacy product from UTI injection solution and Witepsol® S-55. After mixing at 50°C for 0-8 h, UTI suppositories were prepared, which had good UTI content uniformity. Because 2% surfactant was contained in S-55, the UTI injection solution formed a water-in-oil type emulsion as a suppository base. The measured residual moisture content (loss on drying (LOD)) in the prepared vaginal suppositories decreased as the mixing time increased, but their hardness (hardness test (HT)) increased. Near (N) IR spectra of UTI suppositories were measured after mixing for 0-8 h. The best calibration models to predict the HT and LOD of the suppositories were determined based on the NIR spectra by the leave-one-out method in a partial least-squares regression analysis (PLS). The validation result indicated that PLS models for HT and LOD were obtained based on the spectra treated by a combination of smoothing and normalized, respectively, and the model consisted of three latent variables. The plots between the predicted and measured pharmaceutical properties (HT and LOD) based on the calibration data were superimposed with those of the external validation data. The developed NIR spectroscopy method was applied to the preparation process monitoring for UTI vaginal suppositories. In the prepared vaginal suppositories, the predicted LOD decreased as the mixing time increased, and the measured LOD values superimposed well with the predicted values. In contrast, the predicted HT increased as the mixing time increased, and the measured values superimposed with the predicted values.

Differential response properties of peripherally and cortically evoked swallows by electrical stimulation in anesthetized rats.

We compared onset latency, motor-response patterns, and the effect of electrical stimulation of the cortical masticatory area between peripherally and cortically evoked swallows by electrical stimulation in anesthetized rats. The number of swallows and the motor patterns were determined using electromyographic recordings from the thyrohyoid, digastric, and masseter muscles. The onset latency of the first swallow evoked by electrical stimulation of the cortical swallowing area (Cx) was significantly longer than that evoked by stimulation of the superior laryngeal nerve (SLN). The duration of thyrohyoid burst activity associated with SLN-evoked swallows was significantly longer than that associated with either Cx-evoked or spontaneous swallows. Combining Cx with SLN stimulation increased the number of swallows at low levels of SLN stimulation. Finally, A-area (the orofacial motor cortex) stimulation inhibited Cx-evoked swallows significantly more than it inhibited SLN-evoked swallows. These findings suggest that peripherally and cortically evoked swallows have different response properties and are affected differently by the mastication network.

Contribution of synovial lining cells to synovial vascularization of the rat temporomandibular joint.

The lining layer of the synovial membrane in the temporomandibular joint (TMJ) contains two types of lining cells: macrophage-like type A and fibroblast-like type B cells. The type B cells are particularly heterogeneous in their morphology and immunoreactivity, so that details of their functions remain unclear. Some of the type B cells exhibit certain resemblances in their ultrastructure to those of an activated capillary pericyte at the initial stage of the angiogenesis. The articular surface, composed of cartilage and the disc in the TMJ, has few vasculatures, whereas the synovial lining layer is richly equipped with blood capillaries to produce the constituent of synovial fluid. The present study investigated at both the light and electron microscopic levels the immunocytochemical characteristics of the synovial lining cells in the adult rat TMJ, focusing on their contribution to the synovial vascularization. It also employed an intravascular perfusion with Lycopersicon esculentum (tomato) lectin to identify functional vessels in vivo. Results showed that several type B cells expressed desmin, a muscle-specific intermediate filament which is known as the earliest protein to appear during myogenesis as well as being a marker for the immature capillary pericyte. These desmin-positive type B cells showed immunoreactions for vimentin and pericyte markers (neuron-glial 2; NG2 and PDGFRß) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (αSMA and caldesmon). Immunoreactivity for RECA-1, an endothelial marker, was observed in the macrophage-like type A cells. The arterioles and venules inside the synovial folds extended numerous capillaries with RECA-1-positive endothelial cells and desmin-positive pericytes to distribute densely in the lining layer. The distal portion of these capillaries showing RECA-1-immunoreactivity lacked lectin-staining, indicating a loss of blood-circulation due to sprouting or termination in the lining layer. The desmin-positive type B and RECA-1-positive type A cells attached to this portion of the capillaries. Some capillaries in the lining layer also expressed ninein, a marker for sprouting endothelial cells, called tip cells. Since an activated pericyte, macrophage and tip cell are known to act together at the forefront of the vessel sprout during angiogenesis, the desmin-positive type B cell and RECA-1-positive type A cell might serve as these angiogenic cells in the synovial lining layer. Tomato lectin perfusion following decalcification would be a highly useful tool for research on the vasculature of the mineralized tissue. Use of this technique combined with immunohistochemistry should permit future extensive investigations on the presence of the physiological angiogenesis and on the function of the lining cells in the synovial membrane.

A histologic study of deformation of the mandibular condyle caused by distraction in a rat model.

OBJECTIVE: This study investigated the bone resorption process of the rat mandibular condyle after mandibular distraction. STUDY DESIGN: Male Wistar rats at 10 weeks of age underwent unilateral mandibular distraction at 0.175 mm per 12 hours for 10 days. Histologic and histochemical analyses were performed at postoperative day 1 and weeks 1 and 3. RESULTS: High-resolution computed tomography (micro-CT) observations showed that deformation of the condyle occurred in the anterior region, where a discontinuity of the condylar cartilage layer was found in histologic sections. This destroyed area gathered many osteoclasts. In the central region, disorganization with a thin hypertrophic cell layer was recognizable by day 1 but later thickened. Morphologic recovery of the mandibular condyle could be attained by week 3 in this animal model. CONCLUSIONS: These morphologic findings indicate that rapid deformation of the condyle, with destruction of the cartilage layer and bone resorption, was caused by artificial distraction.

Methylmercury monitoring study in Karakuwacho peninsula area in Japan.

Methylmercury (MeHg) is a worldwide concern owing to its adverse health effects. To explore MeHg exposure burdens and the potential contributing factors in different subpopulations in a peninsula area (Karakuwacho) in Japan, a cross-sectional survey was performed. This study included 189 individuals from 102 families. The geometric means of total hair mercury (THg) were 5.74, 3.78 and 2.37 µg/g for adult males, females and children, respectively, of which 56.5 %, 30.9 % and 12.9 % had hair THg exceeding 5 µg/g, respectively. Tuna and mackerel were the common fish species that were positively correlated with hair THg levels in different subpopulations (standardized coefficient ranged from 0.20 to 0.58, p < 0.05). Frequent consumption of these fish species and a large amount of fish intake are likely major contributors of MeHg exposure in this area. Local-scale risk evaluation and risk communication should be highlighted in future studies.

Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology.

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

Immunoexpression of aquaporin-1 in the rat periodontal ligament during experimental tooth movement.

This study examined the immunoexpression pattern of aquaporin-1 (AQP1), first identified as a water channel protein, in the periodontal ligament of rat molars during experimental tooth movement to clarify its role in periodontal responses in an overloaded model by the insertion of a piece of elastic band. In the control group without any treatment, the cementoblasts and osteogenic cells as well as the vascular endothelial cells showed AQP1 immunoreaction. In the experimental group, hyalinized tissue and intensely AQP1 positive amorphous structures which were identified as degenerated endothelial cells by immunoelectron microscopy, occurred at the compression side on Days 1 and 3. AQP1 immunoreaction came to be stronger in the intact endothelial cells around the hyalinized tissue. The hyalinized tissue had almost disappeared by Day 5 when many macrophages reactive to acid phosphatase activity appeared. The periodontal width on Day 7 became almost the same as that in the control group. These findings indicate that the hyalinized tissue and damaged AQP1 positive endothelial cells are phagocytized by macrophages which have temporally migrated, and suggest that the surviving endothelial cells with intense AQP1 reaction are involved in periodontal regeneration by capillary sprouting.

Construction and characterization of a tissue-engineered oral mucosa equivalent based on a chitosan-fish scale collagen composite.

This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the ß1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.

Alterations in intermediate filaments expression in disc cells from the rat temporomandibular joint following exposure to continuous compressive force.

The articular disc in the temporomandibular joint (TMJ) that serves in load relief and stabilizing in jaw movements is a dense collagenous tissue consisting of extracellular matrices and disc cells. The various morphological configurations of the disc cells have given us diverse names, such as fibroblasts, chondrocyte-like cells and fibrochondrocytes; however, the characteristics of these cells have remained to be elucidated in detail. The disc cells have been reported to exhibit heterogeneous immunoreaction patterns for intermediate filaments including glial fibrillary acidic protein (GFAP), nestin and vimentin in the adult rat TMJ. Because these intermediate filaments accumulate in the disc cells as tooth eruption proceeds during postnatal development, it might be surmised that the expression of these intermediate filaments in the disc cells closely relates to mechanical stress. The present study was therefore undertaken to examine the effect of a continuous compressive force on the immunoexpression of these intermediate filaments and an additional intermediate filament - muscle-specific desmin - in the disc cells of the TMJ disc using a rat experimental model. The rats wore an appliance that exerts a continuous compressive load on the TMJ. The experimental period with the appliance was 5 days as determined by previous studies, after which some experimental animals were allowed to survive another 5 days after removal of the appliance. Histological observations demonstrated that the compressive force provoked a remarkable acellular region and a decrease in the thickness of the condylar cartilage of the mandible, and a sparse collagen fiber distribution in the articular disc. The articular disc showed a significant increase in the number of desmin-positive cells as compared with the controls. In contrast, immunopositive cells for GFAP, nestin and vimentin remained unchanged in number as well as intensity. At 5 days after removal of the appliance, both the disc and cartilage exhibited immunohistological and histological features in a recovery process. These findings indicate that the mature articular cells are capable of producing desmin instead of the other intermediate filaments against mechanical stress. The desmin-positive disc cells lacked α-smooth muscle actin (α-SMA) in this study, even though desmin usually co-exists with α-SMA in the vascular smooth muscle cells or pericytes. Because the precursor of a pericyte has such an immunoexpression pattern during angiogenesis, there is a further possibility that the formation of new vessels commenced in response to the extraordinary compressive force.

Zoledronic acid induces S-phase arrest via a DNA damage response in normal human oral keratinocytes.

OBJECTIVE: This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct. DESIGN: Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 µM ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (γ)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2. RESULTS: ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of γ-H2A.X was also seen. CONCLUSIONS: These results indicate that a 10-µM ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiquitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover.

Phenotypes of articular disc cells in the rat temporomandibular joint as demonstrated by immunohistochemistry for nestin and GFAP.

The articular disc is a dense collagenous tissue containing disc cells that are phenotypically described as chondrocyte-like cells or fibrochondrocytes. Despite the possible existence of these phenotypes in systemic joints, little is known about the detailed classification of the articular disc cells in the temporomandibular joint. In this immunocytochemical study we examined the localization and distribution patterns of nestin and glial fibrillary acidic protein (GFAP) in the articular disc of the rat temporomandibular joint at postnatal day 1, and weeks 1, 2, 4 and 8, based on the status of tooth eruption and occlusion. Nestin and GFAP are intermediate filament proteins whose expression patterns are closely related to cell differentiation and cell migration. Both types of immunopositive cell greatly increased postnatally to a stable level after postnatal week 4, but they showed different distribution patterns and cell morphologies. Nestin-reactive disc cells, which were characterized by a meagre cytoplasm and thin cytoplasmic processes, were scattered in the articular disc, whereas GFAP-positive cells, characterized by broader processes, existed exclusively in the deeper area. In mature discs, the major proportion of articular disc cells exhibited GFAP immunoreactivity. Furthermore, a double-immunostaining demonstrated that the nestin-negative cells, consisting of GFAP-positive and -negative cells, exhibited immunoreactions for heat shock protein 25. These findings indicate that the articular disc cells comprise at least three types in the rat temporomandibular joint and suggest that their expressions closely relate to mechanical loading forces within the joint, including occlusal force, as observed through postnatal development.

A morphological analysis on the osteocytic lacunar canalicular system in bone surrounding dental implants.

Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone.

Detection of dicofol and related pesticides in human breast milk from China, Korea and Japan.

Previously, we demonstrated that the concentrations of DDTs were greater in breast milk collected from Chinese mothers than from Japanese and Korean mothers. To investigate dicofol as a possible source of the DDTs in human breast milk, we collected breast milk samples from 2007 to 2009 in China (Beijing), Korea (Seoul, Busan) and Japan (Sendai, Takarazuka and Takayama). Using these breast milk samples, we quantified the concentrations of dichlorobenzophenone, a pyrolysis product of dicofol (simply referred to as dicofol hereafter), dichlorodiphenyltrichloroethane and its metabolites (DDTs) using GC-MS. Overall, 12 of 14 pooled breast milk samples from 210 mothers contained detectable levels of dicofol (>0.1 ng g⁻¹ lipid). The geometric mean concentration of dicofol in the Japanese breast milk samples was 0.3 ng g⁻¹ lipid and significantly lower than that in Chinese (9.6 ng g⁻¹ lipid) or Korean breast milk samples (1.9 ng g⁻¹ lipid) (p<0.05 for each). Furthermore, the ΣDDT levels in breast milk from China were 10-fold higher than those from Korea and Japan. The present results strongly suggest the presence of extensive emission sources of both dicofol and DDTs in China. However, exposure to dicofol cannot explain the large exposure of Chinese mothers to DDTs because of the trace levels of dicofol in the ΣDDTs. In the present study, dicofol was confirmed to be detectable in human breast milk. This is the first report to identify dicofol in human samples.

Detection of acid-sensing ion channel 3 (ASIC3) in periodontal Ruffini endings of mouse incisors.

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion - which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings.

Confirmation of an association of single-nucleotide polymorphism rs1333040 on 9p21 with familial and sporadic intracranial aneurysms in Japanese patients.

BACKGROUND AND PURPOSE: Genetic factors are important determinants of intracranial aneurysm (IA). Recently, a multinational, genome-wide association study identified 3 loci associated with IA, located on 2q (rs700651), 8q (rs10958409), and 9p (rs1333040 and rs10757278). The aim of this study was to evaluate these associations. METHODS: Familial and sporadic cases were investigated. Familial cases, consisting of 96 subjects with IA, and 46 subjects of unknown status from 31 pedigrees were analyzed with the transmission disequilibrium test and linkage analysis. Associations of single-nucleotide polymorphisms (SNPs) with IA were tested in 419 sporadic IA cases and in 408 control subjects. Sequencing of CDKN2A, CDKN2B, and CDKN2BAS revealed additional SNPs, and their associations with IA were also tested. RESULTS: The transmission disequilibrium test revealed associations of 2 SNPs, rs700651 (P=0.036) and rs1333040 (P=0.002), with familial IA. Analysis of SNPs in sporadic cases revealed an allelic association of rs1333040 with IA (odds ratio=1.28; 95% CI, 1.04-1.57; P=0.02) but failed to show associations of rs10757278 and rs496892 with IA. We sequenced 3 candidate genes; CDKN2A, CDKN2B, and CDKN2BAS. All 6 index cases from IA families had the rs1333040-T allele and SNPs (rs10965215, rs10120688, and rs7341791) in CDKN2BAS. None of these SNPs had linkage disequilibrium with rs1333040 and was associated with IA. CONCLUSIONS: A region between introns 7 and 15 of CDKN2BAS carrying the rs1333040-T allele may confer risk for IA.

Levels of perfluorooctane sulfonate and perfluorooctanoic acid in female serum samples from Japan in 2008, Korea in 1994-2008 and Vietnam in 2007-2008.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have recently received attention owing to their widespread contamination in the environment. One of major manufacturers, 3M Company voluntarily phased out PFOS production in 2002. We measured the PFOS and PFOA concentrations in serum samples from Japan (Sendai, Takayama and Osaka), Korea (Busan and Seoul) and Vietnam (Hanoi) to evaluate the possible effects of the phase-out on the serum levels. There were spatial differences in both the serum PFOS and PFOA concentrations. The serum PFOS concentrations (ngmL(-1)) evaluated as the geometric mean (geometric standard deviation) in 2007-2008 ranged from 4.86 (1.45) in Sendai, Japan, to 9.36 (1.42) in Busan, Korea. The serum PFOA concentrations ranged from 0.575 (2.32) in Hanoi, Vietnam, to 14.2 (1.73) in Osaka, Japan. Historically archived samples collected from Korea in 1994-2008 revealed that the serum PFOA concentrations increased by 1.24-fold in Busan from 2000 to 2008 and 1.41-fold in Seoul from 1994 to 2007. On the other hand, the serum PFOS concentrations did not change from 1994 to 2007/2008. The serum PFOS levels in Japan in 2008 were significantly decreased compared with previously reported values (22.3-66.7% of the values in 2003/2004). However, the serum PFOA levels showed a clear decline from 2003 to 2008 in a high-exposed area, Osaka, but not in low-exposed areas in Japan. The trends toward decreases were not uniformly observed in Asian countries, unlike the case for the United States, suggesting that local factors associated with the production and introduction histories in each country overwhelm the effects of the phase-out.

A rare Asian founder polymorphism of Raptor may explain the high prevalence of Moyamoya disease among East Asians and its low prevalence among Caucasians.

BACKGROUND: In an earlier study, we identified a locus for Moyamoya disease (MMD) on 17q25.3. METHODS: Linkage analysis and fine mapping were conducted for two new families in additional to the previously studied 15 families. Three genes, CARD14, Raptor, and AATK, were selected based on key words, namely, "inflammation", "apoptosis", "proliferation", and "vascular system", for further sequencing. A segregation analysis of 34 pedigrees was performed, followed by a case-control study in Japanese (90 cases vs. 384 controls), Korean (41 cases vs. 223 controls), Chinese (23 cases and 100 controls), and Caucasian (25 cases and 164 controls) populations. RESULTS: Linkage analysis increased the LOD score from 8.07 to 9.67 on 17q25.3. Fine mapping narrowed the linkage signal to a 2.1-Mb region. Sequencing revealed that only one newly identified polymorphism, ss161110142, which was located at position -1480 from the transcription site of the Raptor gene, was common to all four unrelated sequenced familial affected individuals. ss161110142 was then shown to segregate in the 34 pedigrees studied, resulting in a two-point LOD score of 14.2 (P = 3.89 × 10(-8)). Its penetrance was estimated to be 74.0%. Among the Asian populations tested (Japanese, Korean, and Chinese), the rare allele was much more frequent in cases (26, 33, and 4%, respectively) than in controls (1, 1, and 0%, respectively) and was associated with an increased odds ratio of 52.2 (95% confidence interval 27.2-100.2) (P = 2.5 × 10(-49)). This allele was, however, not detected in the Caucasian samples. Its population attributable risk was estimated to be 49% in the Japanese population, 66% in the Korean population, and 9% in the Chinese population. CONCLUSION: ss161110142 may confer susceptibility to MMD among East Asian populations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12199-009-0116-7) contains supplementary material, which is available to authorized users.

Past, present, and future of environmental specimen banks.

Environmental specimen banks are an essential part of the infrastructure of environmental sciences. They have various functions: (1) evaluation of governmental environmental policy-making and regulations; (2) a resource for animal health evaluation; (3) research tools to investigate time trends in ecosystems; (4) detection of newly emerging chemicals in the time trends; (5) validations of computer models for environmental phenomena; (6) source identification of contaminants; (7) a tool for food safety; (8) evaluation of genetic selection pressure due to environmental changes. In this review paper, we present a detailed description of the Kyoto University Human Specimen Bank (history, protocol and questionnaires) and provide brief outlines of other representative environmental specimen banks. We then review two illustrative cases in which environmental specimen banks have unveiled insidious contaminations of polybrominated diphenyl ethers and perfluorooctanoic acids. Finally, we give a perspective of new functions for environmental specimen banks in the next 20 years.

Levels and regional trends of persistent organochlorines and polybrominated diphenyl ethers in Asian breast milk demonstrate POPs signatures unique to individual countries.

Human breast milk samples collected in 2007-2008 from four countries, Vietnam (Hanoi), China (Beijing), Korea (Seoul) and Japan (Sendai, Kyoto and Takayama), were analyzed for persistent organic pollutants (POPs) such as dichlorodiphenyltrichloroethane and its metabolites (DDTs), chlordane-related compounds (CHLs), hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). Comparing with previous surveys, the present study indicates that the DDTs in breast milk from China and Vietnam had gradually decreased during the last decade, but were still 5-10 times higher than those in other nations. The ratios of p,p'-DDE/p,p'-DDT and o,p'-DDT/p,p'-DDT were higher in Beijing than in the other countries, suggesting that there is less fresh intake of commercial DDT products and a possible exposure to dicofol in China. CHL and PCB levels were relatively higher in mothers from Japan, whereas beta-HCH and HCB were more common in Chinese women. In Japan, it is suspected that mothers in the urban/coastal area (Sendai) were more continuously exposed to organochlorine pesticides (OCPs) than mothers in the rural/inland area (Takayama). In addition, OCP levels in primiparae were significantly higher than those in multiparae from Japan and Korea. These indicate that both parity and regional factors are major determinants of the levels of OCPs and PCBs in human milk. On the other hand, higher concentrations of PBDEs were observed in mothers' milk from Korea. The congener was dominated by BDE-47 (43-54%), followed by BDE-153 (23-33%) in all regions except for Beijing where BDE-28 (23%) was relatively abundant. In Japanese breast milk, regional and parity-dependent distributions were not observed for PBDEs. Among PBDE congeners, age-dependency was observed for BDE-153, which was negatively correlated (p<0.05) to the age of mothers in Kyoto (17 participants were housewives), while it increased with age in Sendai (10 participants were clerks). No such correlation was seen for BDE-47, indicating that BDE-47 was ingested and assimilated via different kinetics or routes from BDE-153 in Japan.

Paradoxical increases in serum levels of highly chlorinated PCBs in aged women in clear contrast to robust decreases in dietary intakes from 1980 to 2003 in Japan.

OBJECTIVE: Exposure to polychlorinated biphenyls (PCBs) is considered to have culminated between 1950 and 1970 in Japan, and exposure through diet, the major exposure route, has decreased significantly over the last 10 years. The primary goal of the present study was to investigate the long-term trends and congener profiles of serum and dietary levels of PCBs using historical samples. METHODS: Using banked samples collected in 1980, 1995, and 2003 surveys, we determined the daily intakes and serum concentrations of 13 PCB congeners (#74, #99, #118, #138, #146, #153, #156, #163, #164, #170, #180, #182, and #187) in women. RESULTS: The total daily PCB intake [ng/day, geometric mean (geometric standard deviation)] decreased significantly from 523 (2.5) in 1980 to 63 (3.2) in 2003. The serum total PCB level (ng/g lipid) in women <40 years of age decreased significantly from 185 (1.8) in 1980 to 68 (1.8) in 2003. In contrast, the level in women >50 years of age increased significantly from 125 (1.7) in 1980 to 242 (1.7) in 2003. Specifically, the serum concentrations of hexa (#138, #146, #153, #156, #163, and #164) and hepta (#170, #180, #182, and #187) congeners increased significantly. A comparison of the serum PCB levels of women born from 1940 to 1953 revealed that their serum total PCB level was significantly higher in the 2003 survey [242 (1.7), n = 9] than in the 1995 [128 (2.0), n = 17] surveys. This increase in the total PCB level was attributable to increases in the hepta congener groups. CONCLUSION: Present results suggest a decreased rate of elimination of hepta congeners with aging in females, rather than a birth-generation phenomenon.