Keiji Gamoh

Effect of alcohol addition on the movement of petroleum hydrocarbon fuels in soil

Groundwater contamination by fuel spills from aboveground and underground storage tanks has been of growing concern in recent years. This problem has been magnified by the addition of oxygenates, such as ethanol and methyl-tertiary-butyl ether (MTBE) to fuels to reduce vehicular emissions to the atmosphere. These additives, although beneficial in reducing atmospheric pollution, may, however, increase groundwater contamination due to the co-solvency of petroleum hydrocarbons and by the provision of a preferential substrate for microbial utilisation. With the introduction of ethanol to diesel fuel imminent and the move away from MTBE use in many states of the USA, the environmental implications associated with ethanol additive fuels must be thoroughly investigated. Diesel fuel movement was followed in a 1-m soil column and the effect of ethanol addition to diesel fuel on this movement determined. The addition of 5% ethanol to diesel fuel was found to enhance the downward migration of the diesel fuel components, thus increasing the risk of groundwater contamination. A novel method using soil packed HPLC columns allowed the influence of ethanol on individual aromatic hydrocarbon movement to be studied. The levels of ethanol addition investigated were at the current additive level (approx. 25%) for ethanol additive fuels in Brazil and values above (50%) and below (10%) this level. An aqueous ethanol concentration above 10% was required for any movement to occur. At 25% aqueous ethanol, the majority of hydrocarbons were mobilised and the retention behaviour of the soil column lessened. At 50% aqueous ethanol, all the hydrocarbons were found to move unimpeded through the columns. The retention behaviour of the soil was found to change significantly when both organic matter content and silt/clay content was reduced. Unexpectedly, sandy soil with low organic matter and low silt/clay was found to have a retentive behaviour similar to sandy subsoil with moderate silt/clay, but little organic matter. It was concluded that sand grains might have a more important role in the adsorption of petroleum hydrocarbons than first realised. This method has shown that soil packed HPLC columns can be used to provide a quick estimate of petroleum hydrocarbon, and possibly other organic contaminant, movement in a variety of different soil types.

Determination of traces of natural brassinosteroids as dansylaminophenylboronates by liquid chromatography with fluorimetric detection

Abstract A microanalytical method for the determination of traces of natural brassinosteroids as their dansylaminophenylboronates by liquid chromatography with fluorimetric detection is described. The boronates are easily prepared by the reaction of brassinosteroids with dansylaminophenylboronic acid (DABA), which was newly synthesized as a precolumn fluorogenic reagent for brassinosteroids. The potential of the method is demonstrated by the separation of a standard brassinosteroid mixture and the analysis of a biologically active fraction sample obtained from an extract of Zea mays L. pollen. The detection limit of the DABA derivative was found to be 25 pg for brassinolide, which is superior to that of the phenanthreneboronate derivative.

Ferroceneboronic acid as a derivatization reagent for the determination of brassinosteroids by high-performance liquid chromatography with electrochemical detection.

Abstract A micro-scale method for the determination of brassinosteroids as ferroceneboronates by high-performance liquid chromatography with electrochemical detection has been developed. Ferroceneboronic acid (FBA) proved to be satisfactory for use in the derivatization of brassinosteroids with respect to reactivity and sensitivity. The steroids were readily condensed with FBA under mild conditions to provide the corresponding boronates, exhibiting maximum sensitivity at + 0.6 V vs. a silver-silver chloride reference electrode with a detection limit of 25 pg for brassinolide. The method was successfully applied to the determination of natural brassinosteroids in a plant. Brassinolide, dolichosterone, norcastasterone and castasterone were identified in the pollen of sunflower ( Helianthus annuus L.).

A boronic acid derivative as a highly sensitive fluorescence derivatization reagent for brassinosteroids in liquid chromatography

Abstract A sensitive and reactive derivatization reagent for brassinosteroids, 1-cyanoisoindole- 2-m-phenylboronic acid, is readily prepared from o-phthalaldehyde and m-aminophenylboronic acid in one step. The reaction of brassinosteroids with the reagent is done in acetonitrile in the presence of pyridine at 70°C. The resulting boronate shows a single peak of the theoretical shape on the chromatograms and provides excellent sensitivity. The detection limit of the brassinolide boronate in reverse-phase liquid chromatography is 20 pg.

Characterization of Helicobacter pylori Bacteriophage KHP30

ABSTRACT Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.

Postcolumn liquid chromatographic method for the determination of cyanide with fluorimetric detection

Abstract A postcolumn liquid chromatographic procedure for the determination of cyanide with fluorimetric detection was developed. The method is based on both the separation of cyanide ion in the ion-exclusion mode and the postcolumn fluorescence derivatization of cyanide ion with o-phthalaldehyde or naphthalenedicarboxaldehyde in the presence of amino acid. Various parameters such as the kind of amino acid, pH of the reagent buffer and concentration of the sodium salt of EDTA were optimized to provide quantitative conversion of cyanide ion to the fluorescent derivative.

Stereocontrolled synthesis of withanolide D and related compounds

The synthesis of withanolide D, a withanolide having a hydroxy group at the C-20 position, has been accomplished from pregnenolone. The key reactions are based on successful stereochemical control at the C-22 position involving γ-coupling reaction of a lithium enolate with a protected 20-hydroxy-22-aldehyde, and allyl sulphoxide–sulphenate rearrangement of 2,4-dien-1 -one 6β-sulphoxide to introduce the 4β-hydroxy-2,5-dien-1-one system. The related natural withanolides, physalolactone B, deacetyl-physalolactone B, and 3α,20R-dihydroxy-1-oxowitha-5,24-dienolide were also synthesized via a common intermediate for withanolide D.

Liquid chromatographic assay of brassinosteroids in plants

Abstract The liquid chromatographic assay of brassinosteroids (BRs) in plants as boronate derivatives with UV, fluorimetric and electrochemical detection is described. Several boronic acid derivatives proved to be satisfactory for use in the derivatization of BRs with respect to reactivity and sensitivity. BRs were readily condensed with the boronic acid derivatives under mild conditions to provide the corresponding boronates, which exhibit strong absorption, fluorescence or maximum amperometric sensitivity with detection limits of ca. 20–100 pg, depending on the prelabelling reagents. The method was successfully applied to the determination of natural BRs. BRs were identified in the pollen of broad bean (Vicia faba L.), sunflower (Helianthus annuus L.) and buckwheat (Fagopyrum esculentum Moench). The results demonstrate that the liquid chromatographic microanalytical method is useful for the screening of BRs in the plant kingdom.

Identification of brassinolide and castasterone in the pollen of orange (Citrus sinensis Osbeck) by high-performance liquid chromatography

Abstract Brassinosteroids contained in the pollen of Citrus sinensis Osbeck were investigated. By using a rice lamina inclination test, a highly sensitive and specific bioassay for brassinosteroids, as a guide for purification, the pollen was extracted and the extract was subjected to solvent partitioning and subsequent purification by three chromatographic procedures. Two highly purified bioactive fractions were obtained and they were reacted with dansylaminophenylboronic acid. Each derivatized fraction was then analysed by reversed-phase high-performance liquid chromatography with fluorimetric detection. The bioactive compounds contained in the fraction were identified as brassinolide and castasterone by co-chromatography using authentic samples.

Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage

Bacteriophage (phage)-mediated generalized transduction is expected to contribute to the emergence of drug-resistant staphylococcal clones in various environments. In this study, novel phage S6 was isolated from sewage and used to test generalized transduction in human- and animal-derived staphylococci. Phage S6 was a novel type of giant myophage, which possessed a DNA genome that contained uracil instead of thymine, and it could infect all of the tested staphylococcal species. The phage S6 appeared to be similar to the transducing phage PBS1, which infects Bacillus spp. Moreover, phage S6 facilitated the transduction of a plasmid in Staphylococcus aureus and from S. aureus to non-aureus staphylococcal species, as well as vice versa. Transduction of methicillin resistance also occurred in S. aureus. This is the first report of successful intragenus generalized transduction among staphylococci.