Inhibitory effect of Jeju endemic seaweeds on the production of pro-inflammatory mediators in mouse macrophage cell line RAW 264.7.

Seaweed has been used in traditional cosmetics and as a herbal medicine in treatments for cough, boils, goiters, stomach ailments, and urinary diseases, and for reducing the incidence of tumors, ulcers, and headaches. Despite the fact that seaweeds are frequently used in the practice of human health, little is known about the role of seaweed in the context of inflammation. This study aimed to investigate the influence of Jeju endemic seaweed on a mouse macrophage cell line (RAW 264.7) under the stimulation of lipopolysaccharide (LPS). Ethyl acetate extracts obtained from 14 different kinds of Jeju seaweeds were screened for inhibitory effects on pro-inflammatory mediators. Our results revealed that extracts from five seaweeds, Laurencia okamurae, Grateloupia elliptica, Sargassum thunbergii, Gloiopeltis furcata, and Hizikia fusiformis, were potent inhibitors of the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). Based on these results, the anti-inflammatory effects and low cell toxicity of these seaweed extracts suggest potential therapeutic applications in the regulation of the inflammatory response.

Processing and subcellular localization of ADAM2 in the Macaca fascicularis testis and sperm.

Fertilin, a heterodimeric protein complex composed of ADAM1 and ADAM2 located on the sperm surface, is involved in sperm-egg interaction. In our study, we examined the physiological processing and subcellular localization of M. fascicularis ADAM2 during spermatogenesis in the testis and epididymal tract. M. fascicularis ADAM2 was initially synthesized as a 100 kDa precursor in testicular germ cells. After passing into 50 kDa intermediate form in the epididymal tracts, the precursor form was finally processed into a 47 kDa protein in sperm. We found that M. fascicularis ADAM2 is localized on the sperm surface and contributes to the formation of a candidate fertilin complex. In particular, Far-Western blot analysis revealed that M. fascicularis ADAM2 cystein-rich domain may be related to protein-protein interaction. Therefore, the cystein-rich domain of ADAM2 could provide a mechanism to form a fertilin complex.

Importance of the porcine ADAM3 disintegrin domain in sperm-egg interaction.

In the mouse, ADAM3, a well-characterized testis-specific protein of the A disintegrin and metalloprotease (ADAM) family, has a crucial role in fertilization by mediating sperm binding to the egg zona pellucida. However, little is known about ADAM3 in other species, such as domestic pigs. We have identified porcine ADAM3 and analyzed the protein. RT-PCR and trypsinization of sperm surface proteins revealed that porcine ADAM3 is expressed at high levels in the testis and on the sperm surface. Furthermore, an IVF inhibition assay with a recombinant porcine ADAM3 disintegrin domain showed that treatment of the disintegrin domain effectively prevented pig sperm-egg interactions. In the present study, we demonstrated the presence of ADAM3a and ADAM3b molecules in the pig and examined their roles in fertilization.

A new in vitro assay of cholesterol adsorption by food and microbial polysaccharides.

The adsorption of total cholesterol by polysaccharides was measured in vitro by enzymatic reactions, including the polysaccharide precipitation procedure. Total cholesterol adsorption capacities, in a mixture of polysaccharide and total cholesterol, were compared for apple pectin, gelrite gellan gum, xanthan gum, high-methoxyl pectin, citrus pectin, high-viscous alginate, low-viscous alginate, dextran, and zooglan. Acidic polysaccharides such as pectins, alginate, and xanthan gum at concentrations of 0.1% (wt/vol) were able to adsorb over 90% of the total cholesterol when dissolved in distilled water, sodium acetate buffer (pH 4.6), or sodium phosphate buffer (pH 7.0). However, total cholesterol adsorptions by gellan and zooglan were dependent upon the salt concentration and pH value, which decreased cholesterol adsorption in the following order by degree: distilled water, acidic pH, and alkaline pH. In particular, total cholesterol adsorption of zooglan was greatly decreased by the addition of sodium chloride. With 0.1% (wt/vol) polysaccharide dissolved in distilled water, the adsorption capacities of alginate, pectins, gellan gum, xanthan gum, and zooglan were 2.9, 2.88, 2.5, 2.9, and 2.4 mg/dL, respectively. However, 0.2% of zooglan was able to completely adsorb the cholesterol (3 mg/dL), whereas dextran did not adsorb cholesterol at all, producing no precipitate with hexadecyltrimethylammonium bromide.

Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean.

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.