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We have investigated the local free volume and its thermal fluctuation in an amorphous polymethylsilsesquioxane (PMSQ) consisting only of T3 (CH3SiO3/2) and T2 (CH3Si(OH)O2/2) units using photoisomerization of azobenzene molecularly dispersed in the PMSQ films as a photoprobe over a wide temperature range (4−300 K). Photoisomerization profiles are discussed on the basis of three parameters, i.e., first-order plots, final cis fractions, and three-component analysis, for uncured and silanol-condensation-cured PMSQ's. The final cis fraction in the uncured PMSQ decreases markedly below 250 K, which has never been observed in linear polysiloxanes, MQ resins, or carbon-based polymers. The effect of low-molecular-weight components in the PMSQ was evaluated using fractionated PMSQ's by molecular weight. The effect of hydrogen bonding by a silanol group was also studied by temperature dependence of infrared spectra. These results suggest that the low-molecular-weight components with silanol groups fill local vacant space resulting in the anomalous decrease in the final cis fraction for the uncured PMSQ below 250 K.

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The content of 9 types of isoflavonoids (daidzein, glycitein, genistein, formononetin, biochanin A, coumestrol, daidzin, glycitin and genistin) in 34 domestic or imported raw beans including soybeans, 7 immature beans and 5 bean sprouts consumed in Japan were systematically analyzed. Each isoflavonoid was analyzed in total after acid hydrolysis to the aglycone, and intact individual isoflavonoids were also analyzed without hydrolysis. After the sample clean up, daidzein, glycitein, genistein, formononetin, biochanin A, daidzin, glycitin and genistin were determined by HPLC with a diode array detector and coumestrol was determined by spectrofluorimetry. The content and composition of isoflavonoids varied greatly between soybean sprouts, immature soybeans and mature beans of the same type but of different source. Isoflavonoid content was highest in mature soybeans. The composition of isoflavonoids differed in each growth stage of soybeans. In other beans, the largest content of isoflavonoids was found in mature chickpeas, but this value was less than 1/27 of the isoflavonoid content in mature soybeans. Thus, the contribution of beans other than soybeans to the daily intake of isoflavonoids in a Japanese diet is negligible.Key words ---isoflavonoid, bean, acid hydrolysis, high performance liquid chromatography, diode array detection, spectrofluorometric detection tivities 1) and reported to be protective against cancer, cardiovascular diseases and osteoporosis. [3][4][5][6][7][8][9] Much research has been reported about the content of isoflavonoids in soybeans and soybean-derived processed foods. [10][11][12][13][14][15][16][17][18][19][20][21][22][23] In contrast, there are few reports about the isoflavonoid content in beans other than soybeans. 11,12,18,23) Japanese people are reported to ingest isoflavonoids mainly through the consumption of soybeans and its derived processed foods. 20) Recently, we estimated that the Japanese daily intake of isoflavonoids from soybeans and soybean-based processed foods is 27.80 mg per day (daidzein 12.02 mg, glycitein 2.30 mg and genistein 13.48 mg).24) However, isoflavonoid intake from the consumption of immature beans, sprouts and beans other than soybeans has not been elucidated. Here we have measured the content of isoflavonoids in mature and immature beans and bean sprouts consumed in Japan, and have compared the content and composition variation between different types and different growth stages of the beans. MATERIALS AND METHODSMaterials ---Genistein, formononetin and biochanin A were purchased from Extrasynthèse (Genay, France). Daidzein, daidzin, genistin, glycitein and glycitin were from Fujicco Co., Ltd. (Kobe, Japan). Coumestrol was obtained from Fluka Chemie AG (Bucks, Switzerland). Flavone, 2,6-dit-butyl-4-methylphenol (BHT) and dimethyl sulfoxide (DMSO) were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The chemical structures of these isoflavonoids and flavone are shown in Fig. 1. Acetonitrile, ethanol, n-hexane and methanol were analytical grade...

We examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5' non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent that Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.

MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14 mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. The mrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were crossresistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.Poliovirus is a member of the family Picornaviridae, containing a positive-sense, single-stranded RNA as a viral genome. The genome encodes a single precursor polyprotein which is eventually cleaved to four structural and seven nonstructural proteins (58). The structural capsid proteins of picornaviruses are located at the amino-terminal P1 region of the polyprotein. The remainder contains viral nonstructural proteins, including two proteases (2A pro and 3C pro ), an RNA-dependent RNA polymerase (3D pol ), and several other proteins essential for viral RNA synthesis. In poliovirus-infected cells, nonstructural proteins 2B, 2C, 3A, 3B, and 3D and their precursors are associated with a specific structure of virus-induced cytoplasmic membranous vesicles, the so-called replication complex (13,22), and implicated in viral RNA synthesis together with viral RNA and some cellular proteins (13,14,17,22,44,61,64,65,73).The 2C protein of picornaviruses contains conserved nucleoside triphosphate (NTP)-binding and RNA helicase motifs in its middle region (19,(28)(29)(30)(31). Genetically manipulated mutations in the NTP-binding motif of the poliovirus 2C protein abolished viral replication and RNA synthesis (47, 70). The replication of viruses with such mutations in the NTP-binding motif was poorly complementated in trans (71). These results indicate the functional significance of the NTP-binding motif in viral replication. A recombinant poliovirus 2C (or precursor 2BC) protein fused with maltose-binding protein (MBP), MBP-2C, was expressed in Escherichia coli and...

Plasticizers in Japanese retail foods were determined by gas chromatography/mass spectrometry (GC/MS) (SIM). The plasticizers tested were as follows: dibutyl phthalate, butylbenzyl phthalate, di(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate, di(2-ethylhexyl) adipate, diisononyl adipate (DINA), dialkyl adipate, dibutyl sebacate, Oacetyl tributyl citrate (ATBC) and diacetyllauroyl glycerol (DALG). A total of 93 samples were analyzed. For the analysis, each sample was extracted by a method suitable to its nature and cleaned using Florisil ® and Bondesil PSA ® dual layer columns. The recovery of plasticizers from fortified food samples was 62.0-131.0%, except in the case of DINA. The limit of detection (LOD) was different for each sample species and plasticizers. For example, the LOD for plasticizers in retort-pouched baby food was 0.0004-0.037 µg/g. A retort-pouched baby food sample was found to be contaminated by DEHP at the Japanese tolerable daily intake (TDI) level, 40 µg/kg/day. The source of contamination was presumed to be disposable gloves because the baby food was produced before the prohibition of DEHPcontaining poly vinyl chloride (PVC) gloves by the Japanese government. After that prohibition, products generally contained much lower levels of DEHP. A higher level of DALG was detected in the other baby food samples, although it became clear that DALG did not originate as contamination from plastics but was added as a food additive. ATBC was detected in bottled sake samples at levels of around 3-7 µg/g, having migrated from the gasket of the bottle cap. ATBC and DALG levels in the above foods were quite low compared with their no observed adverse effect level (NOAEL) or guideline levels as food additives.Key words ---plasticizer, food, survey, phthalate, adipate from hospitals were found to contain high levels of di(2-ethylhexyl) phthalate (DEHP). 2,3) The authors ascertained the source of the contamination to be disposable PVC gloves used in cooking.3) DEHP causes liver or testicular damage in rats and mice, and the "no observed adverse effect level" (NOAEL) has been shown to be 3.7 or 14 mg/kg/day. 4,5) In June 2000, the Japanese government set the tolerable daily intake (TDI) level for DEHP to be 40-140 µg/kg/ day, and prohibited the use of DEHP-containing gloves for food contact purposes.6) The authors investigated the DEHP levels in retail packed lunches after the prohibition of PVC gloves and found them to be about 4% of the levels before prohibition. 7)In a previous study, a high DEHP content of 6 µg/ g was also found in retort-pouched baby food, with the source being PVC tubing used in production.2)

It has been reported that gonadotropins promoted phosphorylation of ERK/MAPK in granulosa cells. However, little is known about the effects of gonadotropin on ERK activity in theca cells. This study explores how LH/forskolin controls ERK phosphorylation in cultured bovine theca cells. Effects of ERK on steroidogenesis were also investigated. Phosphorylation of ERK in bovine theca cells was augmented by LH and forskolin in 5 min; it decreased thereafter below basal levels in 20 min. Nevertheless, phosphorylation of the ERK kinase, MEK, was unaffected. Addition of H89 (a protein kinase A inhibitor) significantly reduced the effect of LH/forskolin on ERK phosphorylation. A potent MEK inhibitor PD98059 eliminated ERK phosphorylation and augmented progesterone production concomitantly with the elevation of intracellular steroidogenic acute regulatory protein mRNA in LH/forskolin-stimulated theca cells. In contrast to progesterone production, androgen production was diminished significantly by inhibition of ERK with decreased intracellular P450c17 mRNA levels. Taking these results together, we conclude that LH/cAMP leads to phosphorylation of ERK in a biphasic manner through MEK-independent pathway in bovine theca cells. Protein kinase A-induced phosphatase could possibly contribute to the phosphorylation process. Furthermore, modulation of ERK phosphorylation involves control of thecal steroidogenesis via modulation of the expression of steroidogenic acute regulatory protein and P450c17.