Toll-like receptor (TLR) 2 has recently been associated with cellular responses to numerous microbial products, including LPS and bacterial lipoproteins. However, many preparations of LPS contain low concentrations of highly bioactive contaminants described previously as “endotoxin protein,” suggesting that these contaminants could be responsible for the TLR2-mediated signaling observed upon LPS stimulation. To test this hypothesis, commercial preparations of LPS were subjected to a modified phenol re-extraction protocol to eliminate endotoxin protein. While it did not influence the ability to stimulate cells from wild-type mice, repurification eliminated the ability of LPS to activate cells from C3H/HeJ (Lpsd) mice. Additionally, only cell lines transfected with human TLR4, but not human or murine TLR2, acquired responsiveness to both re-extracted LPS and to a protein-free, synthetic preparation of lipid A. These results suggest that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminating endotoxin protein.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.Lipopolysaccharides (LPS) are among the most potent inflammatory bacterial mediators and have been strongly implicated in the inflammatory response associated with gram-negative sepsis. Most LPS signaling studies have used LPS preparations derived from species within the Enterobacteriaceae, which possess relatively well-conserved lipid A structures (reviewed in reference 36). A convergence of data suggest that these prototypic LPS preparations, when highly purified, elicit LPS responses that are restricted in the use of TLR4 as the principal signal-transducing molecule (reviewed in reference 21), which is strongly supported by the finding that synthetic E. coli lipid A activated Toll-like receptor 4 (TLR4) and not TLR2 transfectants (8). However, the lipid A of nonenterobacterial species, e.g., Porphyromonas gingivalis, which has been implicated in the inflammation associated with chronic periodontitis (reviewed in reference 9), differs both structurally and functionally from enterobacterial lipid A. Specifically, the major species of P. gingivalis lipid A is composed of unique branched fatty acids, with longer carbon chains than in enterobacterial lipid A, the absence of a phosphoryl group at position 4Ј of the nonreducing glucosamine, as well as other modifications ( Fig. 1) (1). Consistent with these structural differences is the finding that P. gingivalis LPS activity is poorly inhibited by polymyxin B (12), which has been postulated to inactivate LPS by binding electrostatically to negatively charged phosphate groups, leading to a subsequent interaction of polymyxin B with the hydrophobic fatty acids (25, 33). Although P. gingivalis-induced signaling was shown some time ago to be CD14 dependent (34), site-specific mutagenesis of CD14 suggests that the substitution of certain charged amino acids differentially affects the abilities of Escherichia coli and P. gingivalis LPS to bind CD14 (4, 5). In addition, binding of P. gingivalis LPS to LPS binding protein has been reported to be 100-fo...

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-7 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. lmmunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-7 lines and of phyB-PHYD+ and phyB-phyD-lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is -80% identical in amino acid sequence t o phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-7 mutation indicates that phyD is not essential in some natural environments. INTRODUCTIONDetermining the structures and functions of plant receptor families are important steps toward understanding the molecular mechanisms of plant responses to both externa1 environmental cues and interna1 developmental signals. Red (R) and far-red (FR) light play important roles as environmental signals in deetiolation responses of dark-grown seedlings or dark-adapted plants, in signaling the proximity of neighboring or canopy vegetation via the WFR ratio of light (the shade avoidance response), and in influencing photoperiodic timing (reviewed in Smith, 1994). The plant WFR photoreceptors, members of the phytochrome family, are chromoproteins with photoreversible activation and limited protein sequence similarity to the sensor domains of two-component signal transducers (Schneider-Poetsch, 1992;Kehoe and Grossman, 1996), but they have no well-defined biochemical mode of action (Millar et al., 1994; Pratt, 1995;Quail et al., 1995). In flowering plants, the family comprises at least three major types, designated phyA, phyB, and phyC. These are Current address: University of Utah School of Medicine, Salt Lake City, UT 84132. *To whom correspondence should be addressed. E-mail ubisrQ rnontana.edu; fax 406-994-31 90. encoded by the PHYA, PHYB, and PHYC genes, which are found in both monocots and dicots (Mathews et al., 1995).In many dicot plants, additional PHY genes, which are most likely the products of recent gene duplications, are present (Mathews et al., 1995;Mathews and Sharrock, 1996). Examples include the independent evolution of PHYB-like pairs of genes in at least three highly divergent plant families, the Cruciferae, Solanaceae, and Umbelliferae (Mathews et al., 1995; Pratt et al., 19954, and expansion of PHYA-like groups of genes in...