Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of ϳ170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59 fyn and phospholipase C␥1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59 fyn , suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.

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Fruit and vegetable simple and polyphenols are potent antioxidants. One of the most effective in terms of free radical scavenging is 3,4-dihydroxyphenyl ethanol or hydroxytyrosol (HT), a simple phenol found predominantly in Olea europea, or the olive plant. HT is most abundant in the aqueous fraction of olive pulp with trace amounts in the olive oil fraction and in the leaves. For these experiments, we evaluated the anti-inflammatory activity of olive vegetation water (OVW), which we showed previously to have potent antioxidant activity. Because some simple phenols and polyphenols with antioxidant activity have shown varying anti-inflammatory activities, we tested OVW and HT for their ability to inhibit the production of tumor necrosis factor-alpha (TNF-alpha), a pivotal cytokine in inflammation. In lipopolysaccharide (LPS)-treated BALB/c mice, a model system of inflammation, OVW at a dose of 125 mg/mouse (500 mg/kg) reduced serum TNF-alpha levels by 95%. In the human monocyte cell line, THP-1, OVW reduced LPS-induced TNF-alpha production by 50% at a concentration of 0.5 g/L (equivalent to approximately 0.03 g/L simple and polyphenols). OVW had no toxic effects in vitro or in vivo. When OVW was combined with glucosamine, a component of proteoglycans and glycoproteins that was shown to decrease inducible nitric oxide synthase production in cultured macrophage cells, the 2 compounds acted synergistically to reduce serum TNF-alpha levels in LPS-treated mice. These findings suggest that a combination of OVW and glucosamine may be an effective therapy for a variety of inflammatory processes, including rheumatoid and osteoarthritis.