Determination of gaseous organic compounds in Hyogo Prefecture, Japan.

The aim of this study was to analyze gaseous organic chemicals (GOCs) of high traffic (Nishinomiya City: 979,987 vehicles/day) and low traffic areas (Miki City: 29,338 vehicles/day) by gas chromatography-mass spectrometry (GC-MS) and to evaluate general environment exposure by PAHs in GOCs. After air sampling using an OMNIPORE membrane filter (< 0.45 microm) and Porapak-QS, sorbents were extracted with solvent (dichloromethane: acetone (4:1 v/v), and analysis was carried out by GC-MS. Oxidative derivatives of diethylbenzene, such as diacethylbenzene and ethylacetophenone, were detected in GOCs. PAHs and phthalates in GOCs were measured. Pyrene, benz[a]anthracene, benzo[a]pyrene and benzo[ghi]perylene level were significantly higher in high traffic areas. The geometric mean of pyrene was 0.76 ng/m3 for low traffic areas and 1.96 ng/m3 for high traffic areas; benz[a]anthracene was found at 0.72 ng/m3 and 1.80 ng/m3 in low and high traffic areas, respectively; benzo[a]pyrene was found at 0.87 ng/m3 and 3.60 ng/m3 in low and high traffic areas, respectively and benzo[ghi]perylene was found at 0.57 ng/m3 and 3.04 ng/m3 in low and high traffic areas, respectively. The bis(2-ethylhexyl) phthalate (DEHP) level was the highest in the detected GOCs. The geometric mean of the DEHP levels in high traffic and low traffic areas were 484.85 and 387.26 ng/m3, respectively. Adult and child DEHP exposure levels were 145.32 and 300.33 ng/kg/day, respectively, in high traffic areas. In low traffic areas, adult and child DEHP exposure levels were 116.18 and 240.10 ng/kg/day, respectively.

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element.

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.

Trial to evaluate effects of ambient particulate matter on health: A preliminary study using two-dimensional gel electrophoresis.

OBJECTIVES: Particulate air pollution is a serious problem all over the world, and the development of a method to evaluate the health effects of ambient particles is necessary. In this study, cells cultured in vitro were exposed to particles sampled at the side of a main road, and their protein expression levels were examined. METHODS: Ambient particles were collected at the side of a main road using a high-volume air sampler. Some of the collected particles (crude particles) were treated with an organic solvent to remove chemical components, and the resulting residues were used as residual particles. Cells from the mouse alveolar epithelial cell line LA-4 were inoculated into tissue-culture dishes at 1.4×10(4)/cm(2), exposed to each type of particle or artificial carbon particles (Printex 90) that were dispersed using an ultrasonic homogenizer by mixing in the medium twice at 24 and 48 hours, and incubated for up to 72 hours after the start of inoculation. After exposure, the number of cells and intracellular dehydrogenase activity were measured. Proteins extracted from the cells were subjected to two-dimensional gel electrophoresis with isoelectric focusing at pHs 4-7 using a 10% acrylamide gel, and their expression levels were analyzed after fluorescent staining. RESULTS: The intracellular dehydrogenase activity of the cells significantly decreased as a result of exposure to the residual (0.70-fold) and crude (0.84-fold) particles compared with that of the control, but it showed no change as a result of exposure to Printex 90. The protein expression levels in the cells exposed to the particles increased or decreased similarly, but different expression levels were also observed. There were differences in the effects observed between the cells exposed to the artificial carbon particles and those exposed to particles collected from ambient air. CONCLUSION: This study indicates that protein expression levels in cells change in response to exposure to particles collected from ambient air. To evaluate the effects of particles on health, it is considered necessary to use particles collected from ambient air.

A PTPN11 gene mutation (Y63C) causing Noonan syndrome is not associated with short stature in general population.

Human growth is a highly complicated process, but it is obviously influenced by a genetic factor. Recent genome-wide linkage analyses suggested some genetic regions underlying stature variations. However, any specific genes underlying stature variations have not been identified. Noonan syndrome (NS) is an autosomal dominant disorder clinically characterized by short stature, minor facial anomalies, and congenital heart defects. Recently, PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) has been identified as a major responsible gene for NS, causing about half of the affected individuals. We herein report a large family demonstrating NS caused by one of the common PTPN11 mutations, c.188 A > G (Y63C). In this family, the patients were apparently healthy, but heterozygosity of the c.188 A > G (Y63C) mutation was related to growth impairment. This finding suggested that PTPN11 genetic variants contribute to adult height in the general population. However, c.188 A > G (Y63C) was not identified in 96 short individuals from the general population of 2,281 healthy adults. Thus, it is unlikely that PTPN11 is one of the genes underlying stature variations in the general population.

Proinsulin lacking the A7-B7 disulfide bond, Ins2Akita, tends to aggregate due to the exposed hydrophobic surface.

A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.

The endoplasmic reticulum stress response is stimulated through the continuous activation of transcription factors ATF6 and XBP1 in Ins2+/Akita pancreatic beta cells.

The dominant C96Y mutation of one of the two murine insulin genes, Ins2, causes diabetes mellitus in 'Akita' mice. Here we established pancreatic islet beta cell lines from heterozygous mice (Ins2+/Akita). Western blot analysis of endoplasmic reticulum (ER) molecular chaperones indicated that Grp78, Grp94 and Orp150 are significantly increased in Ins2+/Akita cells compared with wild-type (Ins2+/+) cells. Reporter gene assays using the human GRP78 promoter with or without the ER stress response element (ERSE) showed that Ins2+/Akita cells exhibit significantly stronger ERSE-dependent transcriptional activity than Ins2+/+ cells. Transient over-expression of the Ins2 C96Y mutant in wild-type beta cells induces a stronger ERSE-dependent stress response than does wild-type Ins2 over-expression. The ERSE-binding transcription factor ATF6 is strongly activated in Ins2+/Akita cells. The activity of a reporter containing the specific binding sequence of another ERSE-binding transcription factor, XBP1, is also enhanced in Ins2+/Akita cells. Levels of active forms of XBP1 mRNA and protein are both markedly elevated in Ins2+/Akita cells. These results indicate that this cell line is subject to continuous ER stress and that the Ins2 C96Y mutation induces the expression of ER chaperones through the activation of ATF6 and XBP1.

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan.

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.

Mutation analysis in PKD1 of Japanese autosomal dominant polycystic kidney disease patients.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder (incidence, 1:1,000). The mutation of PKD1 is thought to account for 85% of ADPKD. Although a considerable number of studies on PKD1 mutation have been published recently, most of them concern Caucasian ADPKD patients. In the present study, we examined PKD1 mutations in Japanese ADPKD patients. Long-range polymerase chain reaction (LR-PCR) with PKD1-specific primers followed by nested PCR was used to analyze the duplicated region of PKD1. Six novel chain-terminating mutations were detected: three nonsense mutations (Q2014X transition in exon 15, Q2969X in exon 24, and E2810X in exon 23), two deletions (2132del29 in exon10 and 7024delAC in exon 15), and one splicing mutation (IVS21-2delAG). There was also one nonconservative missense mutation (T2083I). Two other potentially pathogenic missense mutations (G2814R and L2816P) were on the downstream site of one nonsense mutation. These three mutations and a following polymorphism (8662C>T) were probably the result of gene conversion from one of the homologous genes to PKD1. Six other polymorphisms were found. Most PKD1 mutations in Japanese ADPKD patients were novel and definitely pathogenic. One pedigree did not link to either PKD1 or PKD2.

Genetic epidemiology of hereditary hemorrhagic telangiectasia in a local community in the northern part of Japan.

Hereditary hemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by aberrant vascular development. We report here a genetic epidemiologic study in a county, A, in the Akita prefecture (population 1.2 million) located in northern Japan. Nine HHT patients who had been referred to tertiary-care hospitals were located in and near the study county. A total of 137 pedigree members were traced of which 81 were alive and 32 were affected by HHT. Complications associated with cerebral or pulmonary arteriovenous malformations were proven in six out of seven families. Linkage analysis in two large families revealed a weak yet suggestive linkage to the HHT1 locus (encoding endoglin; ENG). Three novel mutations were found in four families, all of which led to a frameshift: a G to C transversion at the splicing donor site of intron 3 (Inv3+1 G>C) in one family, one base pair insertion (A) at nucleotide 828 (exon 7) of the endoglin cDNA in two large families (c.828-829 ins A), and a four base pair deletion (AAAG) beginning with nucleotide 1120 (exon 8) of the endoglin cDNA (c.1120-1123 delAAAG) in one family. The insertion of A in exon 11 (c.1470-1471 insA) mutation found in one family has also been reported in a European family. No endoglin gene mutations were found in two families. The population prevalence of HHT in the county was estimated to be 1:8,000 approximately 1:5,000, roughly comparable with those reported in European and U.S. populations, which is contradictory to the traditional view that HHT is rare among Asians. We recommend that families with HHT be screened for gene mutations in order that high-risk individuals receive early diagnosis and treatment initiation that will substantially alter their clinical course and prognosis.