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Solid-phase microextraction and gas chromatography-mass spectrometry for the determination of polycyclic aromatic hydrocarbons in environmental solid matrices.

A solid-phase microextraction (SPME) and gas chromatography-mass spectrometry method for determining polycyclic aromatic hydrocarbons (PAHs) in environmental solid matrices is developed. Investigated matrices include seaweed (Undaria pinnatifida and Himanthalia elongata), humic substances (isolated from a wetland out-flow and purchased from Aldrich), and soil. Optimal conditions for a good SPME efficiency of 16 hydrocarbon compounds are obtained using a 100- micro m polydimethylsiloxane fiber directly immersed in aqueous carrier medium. The method is remarkable for presenting short extraction times and considerably high sensitivities. The SPME results obtained by using internal calibration give the total analyte concentration based on the identical partitioning behavior of native and spiked pollutants. The detection limits range from 0.001 to 0.1 mg of PAH per kilogram of dry matrix. SPME external calibration provides information regarding freely dissolved analytes. The detection limits range from 0.001 to 0.05 micro g of PAH per liter of carrier medium. The SPME with external calibration procedure can be applied to measure free concentrations of a target compound spiked into a carrier medium and onto a matrix. Based on a comparison of results obtained for the two samples, the partitioning of the target analyte between the matrix and the carrier medium is calculated.

Calibration↗

Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107.

A marine bacterial strain that decomposes the cell walls of some seaweeds, including a Laminaria sp. and Undaria pinnatifida, has been isolated from seawater. This strain has been classified to the genus Vibrio. One of the enzymes which the bacteria secreted into the culture medium was isolated and purified 45-fold from the culture fluid by a combination of ammonium sulfate precipitation and successive rounds of anion-exchange column chromatography. Purified protein migrated as a single band (M(r), 107,000) on sodium dodecyl sulfate-polyacrylamide gels. By amino acid sequence analysis, it was determined that this protein had a single N-terminal sequence that did not exhibit identity with the sequences of other agarases from marine bacteria. This novel enzyme was found to be an endo-type beta-agarase (EC 3.2.1.81) which hydrolyzes the beta-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopy ranosyl (1-->3)-D-galactose] and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose] at a pH of around 8. The optimum temperature was 30 degrees C. This enzyme did not decompose sodium alginate or lambda-, iota-, or kappa-carrageenan. This enzyme may be of practical application in gene technology in the isolation of DNA fragments from agarose gels after electrophoresis.

Amino Acid Sequence↗

Homeostasis as regulated by activated macrophage. II. LPS of plant origin other than wheat flour and their concomitant bacteria.

In order to seek a macrophage-activating substance, lipopolysaccharide (LPS) of plant origin other than that of wheat flour was surveyed. A large amount of LPS (10-100 micrograms/g) was detected in Laminaria japonica (kelp), Curcuma longa (turmeric), Undaria pinnatifida and other substances. Since concomitant bacteria possibly existing in root of farm products can be considered to contribute to LPS of plant origin, a count was taken of bacterial cells both dead and alive. This count revealed that some LPS were derived from concomitant bacteria which had probably come from root. Few concomitant bacterial cells were found in seaweed, while stem-root contained enough bacterial cells. Three predominant bacteria have been isolated and identified; Pantoea agglomerans, Enterobacter cloacae, and Serratia ficaria. These LPSs were purified and their chemical compositions were examined. They are similar to that of Escherichia coli except that their molecular sizes are smaller. Since LPS is non-toxic when taken orally or percutaneously, these LPSs may also be advantageous in the cure of intractable diseases.

Animals↗

Inhibition of icosanoid production in MC/9 mouse mast cells by n-3 polyunsaturated fatty acids isolated from edible marine algae.

The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3 = 18:4n-3 = 18:3n-3 > 20:5n-3 = 16:4n-3 for LTB4; 22:6n-3 = 18:4n-3 = 18:3n-3 > 16:4n-3 > 20:5n-3 (no suppression) for LTC4; 22:6n-3 = 18:4n-3 > 18:3n-3 > 20:5n-3 = 16:4n-3 for 5-HETE.

Animals↗

Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed.

The radical scavenging activity of Japanese edible seaweeds was screened by the DPPH (1-diphenyl-2-picrylhydrazyl) assay to evaluate the DPPH radical scavenging activity in organic extracts. The fresh brown alga Hijikia fusiformis showed the strongest DPPH radical scavenging activity, followed by Undaria pinnatifida and Sargassum fulvellum. The major active compound from Hijikia fusiformis in its acetone extract was identified as fucoxanthin by 13C-NMR spectroscopy.

Antioxidants↗

Purification of stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) from algal fatty acid with lipase and medium pressure liquid chromatography.

Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity.

Chlorophyta↗

Content and characteristics of vitamin B12 in some seaweeds.

The vitamin B12 (B12) content in seven species of seaweed that are consumed frequently in Hokkaido, Japan, was microbiologically measured using Escherichia coli 215. Asakusanori (Porphyra tenera), maruba-amanori (Porphyra suborbiculata) and akaba-gin-nansou (rhodo-glossum pulcherum) showed higher B12 content than the other species, although the content varied greatly among samples in the same species. A bioautography on a thin-layer plate holding a mixture of silica gel and cellulose, differentiation of B12 and its analogues using a binding specificity of intrinsic factor and haptocorrin, and comparison of the B12 concentration determined by the radioisotope dilution assay method using the intrinsic factor as the B12-binding protein with that by the bioassay method, predominantly showed B12 in maruba-amanori and B12 analogues in wakame (Undaria pinnatifida) and akaba-gin-nansou. The B12 uptake of akaba-gin-nansou from artificial seawater was similar to that of asakusanori that contained only B12.

Chromatography, Thin Layer↗

Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-L1 cells.

Fucoxanthin is a major carotenoid found in edible seaweed such as Undaria pinnatifida and Hijikia fusiformis. We investigated the suppressive effects of fucoxanthin and its metabolite, fucoxanthinol, on the differentiation of 3T3-L1 preadipocytes to adipocytes. Fucoxanthin inhibited intercellular lipid accumulation during adipocyte differentiation of 3T3-L1 cells. Furthermore, fucoxanthin was converted to fucoxanthinol in 3T3-L1 cells. Fucoxanthinol also exhibited suppressive effects on lipid accumulation and decreased glycerol-3-phosphate dehydrogenase activity, an indicator of adipocyte differentiation. The suppressive effect of fucoxanthinol was stronger than that of fucoxanthin. In addition, in 3T3-L1 cells treated with fucoxanthin and fucoxanthinol, peroxisome proliferator-activated receptor gamma (PPARgamma), which regulates adipogenic gene expression, was down-regulated in a dose-dependent manner. These results suggest that fucoxanthin and fucoxanthinol inhibit the adipocyte differentiation of 3T3-L1 cells through down-regulation of PPARgamma. Fucoxanthinol had stronger suppressive effects than fucoxanthin on adipocyte differentiation in 3T3-L1 cells.

3T3-L1 Cells↗

Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64.

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.

Amino Acid Sequence↗

Algae -- a poor man's HAART?

Drawing inferences from epidemiologic studies of HIV/AIDS and in vivo and in vitro HIV inhibition by algae, we propose algal consumption as one unifying characteristic of countries with anomalously low rates. HIV/AIDS incidence and prevalence in Eastern Asia ( approximately 1/10000 adults in Japan and Korea), compared to Africa ( approximately 1/10 adults), strongly suggest that differences in IV drug use and sexual behavior are insufficient to explain the 1000-fold variation. Even in Africa, AIDS/HIV rates vary. Chad has consistently reported low rates of HIV/AIDS (2-4/100). Possibly not coincidentally, most people in Japan and Korea eat seaweed daily and the Kanemba, one of the major tribal groups in Chad, eat a blue green alga (Spirulina) daily. Average daily algae consumption in Asia and Africa ranges between 1 and 2 tablespoons (3-13 g). Regular consumption of dietary algae might help prevent HIV infection and suppress viral load among those infected.

Acquired Immunodeficiency Syndrome↗