PP083. Clinic and out-of-clinic blood pressure changes during pregnancy by parity: Boshi study.

INTRODUCTION: Nulliparity is believed to be one of the risk factors for hypertension during pregnancy. However, the relationship between parity and out-of-clinic blood pressure during pregnancy is still unknown. OBJECTIVES: The aim of this study was to evaluate clinic blood pressure and blood pressure measured at home during pregnancy among nulliparous and multiparous women. METHODS: This study was a prospective cohort study. We examined blood pressure measured in the clinic and at home among 530 normotensive pregnant women who received antenatal care at a maternity hospital in Japan. Clinic blood pressures were obtained by duplicate measurements at each antenatal care visit. The participants were also required to measure their own blood pressures every morning at home while they were pregnant. A linear mixed model was used for analysis of the blood pressure course throughout pregnancy [1]. The SAS package (version 9.2) was used for the statistical analyses. RESULTS: A total of 315 nulliparous and 215 multiparous women were entered into this study (mean ages 30.1±4.6years and 33.0±4.1years, respectively). Clinic blood pressure during pregnancy among nulliparous women was significantly higher than that among multiparous women (P=0.02/P<0.0001 for systolic/diastolic blood pressure), whereas there were no significant differences in blood pressure measured at home during pregnancy between them (P=0.42/P=0.22 for systolic/diastolic blood pressure). CONCLUSION: Out-of-clinic blood pressure levels during pregnancy have been shown not to differ between nulliparous and multiparous women, while clinic blood pressure during pregnancy among nulliparous women is higher than that among multiparous women.

Polybrominated diphenyl ethers in human serum and sperm quality.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants; currently, they are identified as ubiquitous environmental contaminants. Several studies indicate that PBDEs might affect male fertility. We present the results of a pilot study on the relationship between human serum PBDEs and sperm quality. The PBDE levels in Japan are comparable to those found in European countries. Strong inverse correlations were observed between the serum concentration of 2,2',4,4',5,5'-hexabromodiphenyl ether and sperm concentration (r = -0.841, p = 0.002) and testis size (r = -0.764, p = 0.01). Extensive studies on the relationship between PBDEs and sperm quality are required.

Dietary intake estimations of polybrominated diphenyl ethers (PBDEs) based on a total diet study in Osaka, Japan.

This study presents the results of a total diet study performed for estimating the dietary intake of polybrominated diphenyl ethers (PBDEs) in Osaka, Japan. The concentrations of 36 PBDEs were measured in samples from 14 food groups (Groups I-XIV). PBDEs were detected only in Groups IV (oils and fats), V (legumes and their products), X (fish, shellfish, and their products), and XI (meat and eggs) at concentrations of 1.8, 0.03, 0.48, and 0.01 ng g⁻¹, respectively. For an average person, the lower bound dietary intakes of penta- and deca-formulations were estimated to be 46 and 21 ng day⁻¹, respectively. A high proportion of the decabrominated congener (DeBDE-209) was observed in Group IV. To confirm the presence of DeBDE-209 in vegetable oils, an additional analysis was performed using 18 vegetable oil samples. Of these, seven contained ng g⁻¹ levels of DeBDE-209.

Molecular analysis of the genome segments S1, S4, S6, S7 and S12 of a Rice gall dwarf virus isolate from Thailand; completion of the genomic sequence.

The complete nucleotide sequences of the double-stranded RNA segments S1, S4, S6, S7 and S12 of the genome of a Rice gall dwarf virus (RGDV) isolate from Thailand were determined. The segments consisted of 4505, 2622, 1648, 1652 and 853 nucleotides, encoding putative proteins of 1458, 725, 489, 511 and 206 amino acids with molecular masses of approximately 166, 80, 53, 59 and 24 kDa, respectively. Homology searches indicated that each of the putative proteins has a counterpart in isolates of Rice dwarf virus (RDV) and Wound tumor virus, two other species in the genus Phytoreovirus. However, no similarities were found to other registered sequences, including those of other viruses that belong to the family Reoviridae. The identities between homologous structural proteins of RGDV and RDV ranged from 34 to 51% and were thus higher than those between homologous non-structural proteins of RGDV and RDV (16-37%). Among the nonstructural proteins, the highest amino acid sequence identity (37%) was observed for RGDV Pns11 and RDV Pns10, a constituent of tubular inclusions. This observation suggests that a specific amino acid backbone might be required for maintaining not only the three-dimensional structure of virions but also that of inclusions. The entire sequence of the RGDV genome is now available.

High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome.

To clarify some characteristics of phosphatidylinositol glycan-class A gene (PIG-A) mutations in aplastic anemia (AA) and myelodysplastic syndrome (MDS) patients compared with those in paroxysmal nocturnal hemoglobinuria (PNH) patients, we investigated PIG-A mutations in CD59- granulocytes and CD48- monocytes from seven AA, eight MDS, and 11 PNH Japanese patients. The most frequent base or type abnormalities of the PIG-A gene in AA and MDS patients were base substitutions or missense mutations, respectively, and deletions or frameshift mutations, respectively, in PNH patients. Several PIG-A mutations, most of which were statistically minor, were found in glycosylphosphatidylinositol-negative cells from all AA and MDS patients but not from all PNH patients. However, the common PIG-A mutations during the clinical course between CD59- granulocytes and/or CD48- monocytes from each AA or MDS patient, except for Case 5, were not found. PIG-A mutations were different between the granulocytes and monocytes from five AA and five MDS patients. Our results indicate that there were some characteristics of PIG-A mutations in AA and MDS patients compared with PNH patients and that several minor PNH clones in these patients occurred at random during the clinical course. This partly explains the transformation of AA or MDS to PNH at intervals.

A novel ABC transporter gene, PMR5, is involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.

We have cloned a novel ABC transporter gene PMR5 from the phytopathogenic fungus Penicillium digitatum by RT-PCR using degenerate primers. The deduced amino acid sequence of PMR5 showed 37% identity to PMR1 from the same fungus, 71% identity to AtrB from Aspergillus nidulans, and 65% identity to BcatrB from Botrytis cinerea. Disruption mutants for PMR5 were generated in two independent P. digitatum strains and their phenotypes were characterized. These mutants displayed increased sensitivity to thiabendazole (a benzimidazole), benomyl (a benzimidazole), dithianon (a quinone), resveratrol (the phytoalexin of grape), and camptothecin (an alkaloid). Delta pmr1 disruption mutants were previously reported to show resistance to demethylation inhibitors (DMIs). These mutants were found also to display increased sensitivity to phloretin (the phytoanticipin of apples), camptothecin and oligomycin (an antibiotic). Transcription of PMR1 and PMR5 was strongly induced in response to several toxicants, including DMIs that specifically induced PMR1. In contrast, dithianon and resveratrol specifically induced PMR5 transcription. These findings indicate that expression of the two ABC transporter genes is regulated differently, and that they have complementary roles in multidrug resistance, with each having different substrate-specificities.

Multiresidue analysis of pesticides in vegetables and fruits using two-layered column with graphitized carbon and water absorbent polymer.

A high-throughput multiresidue analysis of pesticides in non-fatty vegetables and fruits was developed. The method consisted of a single extraction and a single clean-up procedure. Food samples were extracted with ethyl acetate and the mixture of extract and food dregs were poured directly into the clean-up column. The clean-up column consisted of two layers of water-absorbent polymer (upper) and graphitized carbon (lower), which were packed in a reservoir (75 ml ) of a cartridge column. The polymer removed water in the extract while the carbon performed clean-up. In a recovery test, 110 pesticides were spiked and average recoveries were more than 95% from spinach and orange. Most pesticides were recovered in the range 70-115% with RSD usually < 10% for five experiments. The residue analyses were performed by the extraction of 12 pesticides from 13 samples. The two methods resulted in similar residue levels except chlorothalonil in celery, for which the result was lower with the proposed method. The results confirmed that the proposed method could be applied to monitoring of pesticide residue in foods.

PCR-based detection of sterol demethylation inhibitor-resistant strains of Penicillium digitatum.

A simple method for detecting sterol demethylation inhibitor (DMI)-resistant strains of the citrus green mould pathogen, Penicillium digitatum, has been developed. The method involves detection of a tandem repeat of a transcriptional enhancer in the promoter region of PdCYP51, which encodes the target enzyme of DMIs, by PCR, using conidia as template. The presence of the tandem repeat leads to overexpression of this gene and confers DMI resistance to the fungus. We examined the relationship between the presence of the tandem repeat and DMI resistance in 39 strains of P digitatum. The results suggested that the DMI resistance mechanism based on the tandem repeat is common in this fungus. Using this method, the presence of DMI resistance of this fungus can be detected within 5-6 h.

[Drying ability of anhydrous sodium sulfate on wet organic solvents after liquid-liquid partition].

Water concentration in organic solvents after liquid-liquid partition was determined by the Karl Fischer titration method. n-Hexane and petroleum ether showed quite low levels of water, such as 0.1 mg/mL. The water concentration in wet ethyl acetate was about 20-30 mg/mL and that in diethyl ether was about 8-10 mg/mL. Anhydrous sodium sulfate absorbed about 20-25% of the water after vigorous mixing with wet ethyl acetate or diethyl ether. Wet acetonitrile extract from wet food, which contained about 60 mg/mL water after salting out with sodium chloride, was not dried at all with anhyfrous sodium sulfate treatment. Spiking n-hexane into wet ethyl acetate or wet diethyl ether was effective to exclude water. Spiking toluene into salted acetonitrile drove out water and dissolved sodium chloride. It can be concluded that the drying ability of anhydrous sodium sulfate towards wet organic solvents is poor, but it is effective in removing suspended water in solvents.

GC/MS analysis of polybrominated diphenyl ethers in fish collected from the Inland Sea of Seto, Japan.

Development of an analytical method for polybrominated diphenyl ethers (PBDEs) in fish and their concentration in Japanese marine fish were investigated. Fish homogenate was extracted with diethyl ether/hexane (1 + 3). The extract was cleaned up by automated gel permeation chromatography (GPC) and then by mini-column chromatography, which consisted of three layers of silica gel and sulfuric acid-impregnated silica gel. The PBDE fraction was concentrated and injected into a GC/MS with negative chemical ionization (NCl). Recoveries of the 15 individual PBDEs (BDE-15, 28, 37, 47, 66, 71, 75, 77, 85, 99, 100, 119, 153, 154, and 209) each at a fortification level of 4 ng/g lipid were in the range of 88-128% and the relative standard deviations (RSD) were 0.43-7.6% (n = 4). Seven species of marine fish (conger eel, flounder, gray mullet, horse mackerel, red sea bream, sea bass, and yellowtail) were collected from the Inland Sea of Seto, and were analyzed with the developed method. Seven PBDEs (BDE-28, 47, 66, 99, 100, 153, and 154) were detected in all the samples. The most abundant PBDE congener was BDE-47 found in all the samples. Relatively high levels of PBDEs were found in the gray mullets and yellowtails.

Tandem repeat of a transcriptional enhancer upstream of the sterol 14alpha-demethylase gene (CYP51) in Penicillium digitatum.

We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450(14DM)) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.

Sequence analysis of Pns11, a nonstructural protein of rice gall dwarf virus, and its expression and detection in infected rice plants and vector insects.

The nucleotide sequence of genome segment S11 of rice gall dwarf virus (RGDV), a member of Phytoreovirus, was determined. The segment encodes a putative protein of 40 kDa that exhibits approximately 37% homology at the amino acid level to the nonstructural proteins Pns10 of rice dwarf and wound tumor viruses, which are other members of Phytoreovirus. A band of a protein with an apparent molecular mass of 40 kDa was specifically detected in an analysis of cells transfected with S11 cDNA. An antiserum raised against this protein reacted with a protein of approximately 40kDa after fractionation by SDS-PAGE of materials prepared from infected plants and from viruliferous vector insects. However, the antiserum did not react with purified viral proteins. These results suggest that S11 encodes a nonstructural protein of RGDV. This protein was named Pns11.

Determination of triazine herbicides in foods with liquid chromatography mass spectrometry.

Eight residual triazine herbicides and three metribuzin metabolites in foods were determined by liquid chromatography mass spectrometry (LC-MS) with an atmospheric pressure chemical ionization (APCI) interface, under both positive and negative ion modes. Herbicides were extracted with acetonitrile, and no cleanup procedure was adopted in this method. Four foods were spiked with eight herbicides and three metabolites at 0.05 ppm. The average recoveries of these herbicides usually ranged from 82 to 99% and the relative standard deviations were usually around 10%. These results suggest that LC-MS with APCI can be used to determine residues of triazine herbicides in foods.

Biological Control of Cyclamen Soilborne Diseases by Serratia marcescens Strain B2.

Cyclamen plants were treated with a highly chitinolytic bacterium, Serratia marcescens strain B2, and then challenge inoculated with Rhizoctonia solani sclerotia or Fusarium oxysporum f. sp. cyclaminis conidia. The bacterium suppressed these fungal diseases of cyclamen plants, especially the damping off caused by R. solani, in a greenhouse. Strain B2 survived at approximately 106 to 107 CFU/g in soil for 4 months after the initial application under greenhouse conditions. Chitinolytic enzymes and antifungal low-molecular-weight compounds were present in filtrates of S. marcescens B2, which suppressed germination of R. solani sclerotia in vitro.

Multiresidue analysis of pesticides in vegetables and fruits using a high capacity absorbent polymer for water.

A single extraction and a single clean-up procedure was developed for multi-residue analysis of pesticides in non-fatty vegetables and fruits. The method involves the use of a high capacity absorbent polymer for water as a drying agent in extraction from wet food samples and of a graphitized carbon column for clean-up. A homogeneously chopped food sample (20 g) and polymer (3 g) were mixed to absorb water from the sample and then 10 min later the mixture was vigorously extracted with ethyl acetate (100 ml). The extract (50 ml), separated by filtration, was loaded on a graphitized carbon column without concentration. Additional ethyl acetate (50 ml) was also eluted and both eluates were concentrated to 5 ml for analysis. The procedure for sample preparation was completed within 2 h. In a recovery test, 107 pesticides were spiked and average recoveries were more than 80% from asparagus, orange, potato and strawberry. Most pesticides were recovered in the range 70-120% with usually less than a 10% RSD for six experiments. The results indicated that a single extraction with ethyl acetate in the presence of polymer can be applied to the monitoring of pesticide residues in foods.

A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.

Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Deltapmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.

The complete nucleotide sequence of the rice grassy stunt virus genome and genomic comparisons with viruses of the genus Tenuivirus.

Rice grassy stunt virus (RGSV, IRRI isolate) has six genomic RNA segments. The nucleotide (nt) sequences of RNAs 1-4 were determined. The cumulative length of the RGSV genome, including RNAs 5 and 6, was 25142 nt. All six RNA segments had an ambisense coding strategy and almost identical terminal sequences over 17 nt. The virus complementary (vc) sequence of the largest segment, RNA1, had an open reading frame encoding a protein of Mr 339133 (the 339.1K protein), while the virus sense (v) sequence encoded a protein of Mr 18910 in the 5'-proximal region. The predicted 339.1K protein contained the highly conserved motifs of the RNA-dependent RNA polymerase and a short but distinct Arg/Gly-rich stretch at the C terminus. The putative RNA polymerase showed strong similarity with that of rice stripe tenuivirus (RSV); they shared 37.9% amino acid identity over 2140 residues. The predicted proteins of Mr 23280 on vRNA2 and 93 879 on vcRNA2 were only slightly similar in sequence to the proteins encoded by vRNA2 and vcRNA2 of other tenuiviruses. The predicted proteins encoded by RNA3 and RNA4 did not show significant similarity to any database proteins. Only the putative RNA polymerase encoded on RNA1 was well-conserved between RGSV and RSV. The low sequence similarities in proteins encoded by RNAs 2, 5 and 6, together with the unique RNA segments 3 and 4, indicate that RGSV may be distinct from other tenuiviruses.

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea).

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance.

Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme.

The human lysozyme gene, which is assembled by the stepwise ligation of chemically synthesized oligonucleotides, was introduced into tobacco (Nicotiana tabacum cv `SR1') by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed under the control of the cauliflower mosaic virus 35S promoter, and the gene product accumulated in the transgenic tobacco plants. The transgenic tobacco plants showed enhanced resistance against the fungus Erysiphe cichoracearum - both conidia formation and mycelial growth were reduced, and the size of the colony was diminished. Microscopic observation revealed that the transgenic tobacco plants carried the resistant phenotype, analogous to that of the resistant cultivar `Kokubu' which had been selected by conventional breeding. Growth of the phytopathogenic bacterium Pseudomonas syringae pv. tabaci was also strongly retarded in the transgenic tobacco, and the chlorotic halo of the disease symptom was reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases.