The chemokine, CXCL16, and its receptor, CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro.

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.

Increased IL-17 expression in degenerated human discs and increased production in cultured annulus cells exposed to IL-1ß and TNF-α.

IL-17 is expressed in a number of tissues including the intervertebral disc, where it exerts strong inflammatory properties. We evaluated IL-17 using immunolocalization in herniated and non-herniated human discs, IL-17 gene expression, and the production of IL-17 by annulus cells cultured in three dimensions in the presence of IL-1ß or TNF-α. There was no difference in the percentage of IL-17 positive cells in annulus or nucleus in herniated vs. non-herniated disc specimens. Molecular studies confirmed expression of IL-17 in disc tissue, with significantly increased expression in more degenerated discs; there was no difference in expression between herniated vs. non-herniated discs. Exposure to IL-1ß or TNF-α resulted in significantly greater production of IL-17. Our findings expand understanding of IL-17 production by disc cells and reveal the importance of non-canonical IL-17 production in the disc. Significantly greater expression of IL-17 in more degenerated discs adds to our understanding of the changes in disc cell function with advancing stages of disc degeneration.

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.

A dual-luciferase reporter system for studying recoding signals.

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.