Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

Activation of endogeneous retroviruses in mouse cells by thermal neutrons

The effect of thermal neutrons on the induction of murine endogenous viruses from a mouse fibroblast cell line was investigated. Thermal neutrons were more effective than X-rays in induction of endogenous virus as well as in killing of the cells. However, when measured as a function of cell killing, both radiations had similar efficiency of induction. The RBEs of thermal neutrons alone were calculated on the assumption that the contribution of contaminating gamma-rays was additive. It was 4.2 for the killing effect and 4-5 for virus induction.     

Paper disk assay for glycosaminoglycan sulfotransferases

A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [35S]sulfate-labeled products are precipitated and then separated from a sulfate donor ([35S]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

The Engelbreth-Holm-Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans

Glycosaminoglycans were prepared from the Engelbreth-Holm-Swarm mouse tumor. Enzymatic analysis demonstrated heparan sulfate (95.8%) and chondroitinase ABC-sensitive galactosaminoglycans (4.2%). HPLC analysis of the disaccharide units showed that heparan sulfate chains were undersulfated on average, comprising approximately 30% nonsulfated and 60% N-sulfated disaccharide units with small proportions of other monosulfated and disulfated disaccharide units. In contrast, galactosaminoglycan chains were oversulfated, containing an appreciable proportion (15%) of a 4,6-disulfated (so-called E-type) disaccharide unit in addition to 51% of a 4-sulfated, 22% of a 6-sulfated, and 11% of a nonsulfated disaccharide unit. The significance of the oversulfated disaccharide structure is discussed in relation to the possible regulation of functions of hybrid proteoglycans from which the galactosaminoglycan chains are derived.     

Proportionality of ELF electric field-induced growth inhibition to induced membrane potential inZea mays andVicia faba roots

Summary The postulate that 60-Hz electric field-induced bioeffect severity is proportional to induced transmembrane potential [ ] magnitude was tested and supported using a plant root model cell system. Statistically significant correlations were obtained upon regression of the relative rates of exposedVicia faba andZea mays root segment growth on the average (calculated) arising in those segments under specified 60 Hz field exposure conditions. The associated with the apparent threshold for growth inhibition was similar inZea andVicia roots (2.5 vs 2.4 mV, respectively). At greater than this threshold,Zea root growth declined by about 9% per mV, andVicia root growth by about 19% per mV induced potential.Abbreviations EF electric field - RGR relative growth rate - RSGR relative segmental growth rate - induced membrane potential - segmental-average induced membrane potential - VC _d region of root tip in which a complete, nascent vascular cylinder is first distinguishable in histological sections     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze-substitution fixation

We have used the freeze-substitution fixation technique for electron microscopy of yeast cells that express the hepatitis B virus core antigen (HBcAg) following transformation with the cloned gene. Abundant spherical particles were found within the transformed cells. These particles had a uniform size and shape, measured about 21 nm in diameter, had electron-lucent centers, and consisted of many subunits. They were localized in both the cytoplasm and the nucleus. None of these particles was found in the cells of the parent strain. Comparison of the HBcAg particles isolated from the yeast cells and the particles within the yeast cells demonstrated that the 21-nm particles were in fact ultrastructurally superimposable on HBcAg. Thus, the HBcAg particles within the yeast cells were similar to the HBcAg particles in human liver tissues infected with hepatitis B virus, not only in their size and appearance, but also in their intracellular localization. These results suggest that the yeast cell has the same machinery for synthesis and intracellular translocation of the HBcAg polypeptides as the human cell.