**6. Analysis of the evolutionary relationship of vulture TLR and IκBα**

The dendrogram of sequences was calculated based on the distance matrix that was generat‐ ed from the pairwise scores and the phylogenetic trees were constructed based on the multi‐ ple alignment of the sequences using the PHYLIP (Phylogeny Inference Package) available at the expasy.org web page. All ClustalW phylogenetic calculations were based around the neighbor-joining method of Saitou and Nei (Saitou & Nei, 1987).

For the analysis of the evolutionary relationship of vulture and other vertebrate TLR and IκBα, a phylogenetic tree was constructed with the TIR-domain sequences of human, maca‐ que, bovine, pig, mouse, Japanese pufferfish and chicken TLR1. GenBank or swiss protein accessions numbers Q5WA51, Q706D2, Q6A0E8, Q59HI9, Q5H727 and Q5FWG5, respec‐ tively. The phylogenetic analysis of the TIR domain of vulture TLR1 revealed separate clus‐ tering of TLR1 from birds, fish, mouse and other mammals (Fig. 10B)

For the TLRs, it is assumed that the structure of the ectodomain has evolved more quickly than the structure of the TIR (Johnson, 2003). Similarly to other TLR receptors, the degree of homology of vulture TLR1 was higher in the transmembrane and cytoplasmic domains than in the extracellular domain. As expected, phylogenetic analysis of the TIR domains revealed separate clustering of TLR1 from birds, fish and mammals (Fig. 9B), suggesting independent evolution of the Toll family of proteins and of innate immunity (Beutler & Rehli, 2002; Roach et al., 2005).

The unrooted trees were constructed by neighbor-joining analysis of an alignment of the an‐ kirin repeats of IκBα sequences from vulture and other species (A) or the alignment of the TIR domains of TLR1 from vulture and other species (B). The branch lengths are proportion‐ al to the number of amino acid differences. GenBank or swiss protein accessions numbers of chicken (*Gallus gallus*), human (*Homo sapiens*), mouse (*Mus musculus*), rat (*Rattus norvegicus*), African frog (*Xenopus laevis*), cattle (*Bos taurus*), zebrafish (*Danio rerio*), Mongolian gerbil (*Meriones unguiculatus*), Rainbow trout (*Oncorhynchus mykiss*) and pig (*Sus scrofa*) sequences used for the phylogenetic tree were Q91974, P25963, Q08353, Q63746, Q1ET75, Q6DCW3, Q8WNW7, Q6K196, Q1ET75, Q8QFQ0 and Q9Z1E3, respectively.

tion of the innate immune response may vary considerably among species (Rehli, 2002). Consistent with its role in pathogen recognition and host defense, the tissue and cell expres‐ sion pattern of vulture TLR1, as revealed by real time RT-PCR, correlated with vulture abili‐ ty to respond to various pathogenic challenges. The expression of vulture TLR1 was higher in cells such as circulating PBMC and intestinal epithelial cells that are immediately accessi‐

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The analysis of the relative expression of IκBα mRNA transcripts, using real-time RT-PCR, demonstrated that vulture IκBα mRNAs were higher in lung, artery, heart, and in PBMC cells (Fig. 9), which was consistent with its role in numerous physiological processes. Inter‐ estingly, the expression of vulture IκBα mRNA was observed in tissues at which the lowest expression of vulture Toll-like receptor was found. This is consistent with the role of IκBα as

**6. Analysis of the evolutionary relationship of vulture TLR and IκBα**

neighbor-joining method of Saitou and Nei (Saitou & Nei, 1987).

tering of TLR1 from birds, fish, mouse and other mammals (Fig. 10B)

The dendrogram of sequences was calculated based on the distance matrix that was generat‐ ed from the pairwise scores and the phylogenetic trees were constructed based on the multi‐ ple alignment of the sequences using the PHYLIP (Phylogeny Inference Package) available at the expasy.org web page. All ClustalW phylogenetic calculations were based around the

For the analysis of the evolutionary relationship of vulture and other vertebrate TLR and IκBα, a phylogenetic tree was constructed with the TIR-domain sequences of human, maca‐ que, bovine, pig, mouse, Japanese pufferfish and chicken TLR1. GenBank or swiss protein accessions numbers Q5WA51, Q706D2, Q6A0E8, Q59HI9, Q5H727 and Q5FWG5, respec‐ tively. The phylogenetic analysis of the TIR domain of vulture TLR1 revealed separate clus‐

For the TLRs, it is assumed that the structure of the ectodomain has evolved more quickly than the structure of the TIR (Johnson, 2003). Similarly to other TLR receptors, the degree of homology of vulture TLR1 was higher in the transmembrane and cytoplasmic domains than in the extracellular domain. As expected, phylogenetic analysis of the TIR domains revealed separate clustering of TLR1 from birds, fish and mammals (Fig. 9B), suggesting independent evolution of the Toll family of proteins and of innate immunity (Beutler & Rehli, 2002;

The unrooted trees were constructed by neighbor-joining analysis of an alignment of the an‐ kirin repeats of IκBα sequences from vulture and other species (A) or the alignment of the TIR domains of TLR1 from vulture and other species (B). The branch lengths are proportion‐ al to the number of amino acid differences. GenBank or swiss protein accessions numbers of chicken (*Gallus gallus*), human (*Homo sapiens*), mouse (*Mus musculus*), rat (*Rattus norvegicus*), African frog (*Xenopus laevis*), cattle (*Bos taurus*), zebrafish (*Danio rerio*), Mongolian gerbil (*Meriones unguiculatus*), Rainbow trout (*Oncorhynchus mykiss*) and pig (*Sus scrofa*) sequences

ble to microorganisms upon infection.

Applications

258

inhibitor of the TLR-signalling pathway.

Roach et al., 2005).

For the analysis of the evolutionary relationship of vulture and other vertebrate IκBα, a phy‐ logenetic tree was constructed with the sequences of chicken, human, mouse, rat, African frog, cattle, zebrafish, Mongolian gerbil, Rainbow trout and pig IκBα. The phylogenetic analysis of the ankyrin domain of vulture IκBα revealed separate clustering of IκBα from rodents, fish and other species and the sequence of vulture IκBα clustered together with that of chicken IκBα (Fig 10A). The IκB family includes IκBα, IκBβ, IκBγ, IκBε, IκBζ, Bcl-3, the precursors of NFκB1 (p105), and NF-κB2 (p100), and the Drosophila protein Cactus (Hayden et al., 2006; Karin & Ben-Neriah, 2000; Totzke et al., 2006; Gilmore, 2006). Why multiple IκB proteins now exist in vertebrates has been a subject of great interest, and much effort has been expended on establishing the roles of individual members of this protein family in the regulation of NF-κB. The recent identification of a novel member of IκB family (IκBζ) indi‐ cates that there might exist species-specific differences in the regulation of NF-κB (Totzke et al., 2006).

**Figure 10.** Phylogenetic trees illustrating the relationship between TLR and IκBα sequences from vulture and other species.

Evolutionarily, the IκB protein family is quite old, as members have been found in insects, birds and mammals (Ghosh & Kopp, 1998). However, the finding that individual ankyrin repeats within each IκB molecule are more similar to corresponding ankyrin repeats in other IκB family members, rather than to other ankyrin repeats within the same IκB, suggests that all IκB family members evolved from an ancestral IκB molecule (Huguet, et al., 1997).

Consistent with the hypothesis that all these factors evolved from a common ancestral RHDankyrin structure within a unique superfamily, explaining the specificities of interaction be‐ tween the different Rel/NF-kappa B dimers and the various I kappa B inhibitors (Huguet, et al., 1997).

ecules that neutralize toxins found in the genetic and phenotypic background of an organism (like vulture) is extremely adequate for bio compatible drugs and antidote devel‐

Identification of Key Molecules Involved in the Protection of Vultures Against Pathogens and Toxins

http://dx.doi.org/10.5772/54191

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**8. Biotechnological applications of molecules involved in the recognition**

Our growing understanding of host-pathogen interactions and mechanisms of protective immunity have allowed for an increasingly rational approach to the design of immune based therapeutics. One posible biomedical application of the discovery of efficient patho‐ gen receptors could be the generation of "immunoadhesins" (Perez de la Lastra et al., 2009). Because of the versatility of immunoadhesins, immunoadhesin-based therapies could, in theory, be developed against any existing pathogen. Some advantages of immunoadhesinbased therapies include versatility, low toxicity, pathogen specificity, enhancement of im‐ mune function, and favorable pharmacokinetics; the disadvantages include high cost, limited usefulness against mixed infections and the need for early and precise microbiologic

The patent by Visintin *et al*. (cited in Perez de la Lastra et al., 2009) discloses anti-pathogen immunoadhesins (APIs), a subset of which is "tollbodies", which have a pathogen recogni‐ tion module derived from the binding domain of a toll-like receptor (TLR). A schematic il‐

**Figure 11.** Schematic structure of an anti-pathogen immunoadhesin (API). Gray undashed area, pathogen recognition module; dashed, Fc portion. Disulfide bridges are represented by dashed line, which include intrachain bridges (that

APIs can be used as therapeutics, e.g., for treating pathogen-associated disorders, e.g., infec‐ tions and inflammatory conditions (e.g. inflammatory conditions associated with a patho‐

stabilize the Ig domains) and the interchain bridges (that covalently link two immunoadhesin molecules).

lustration of an exemplary API is shown in Fig. 11.

opment.

**of pathogens**

diagnosis.

Recently, the presence of two IkappaB-like genes in Nematostella encoded by loci distinct from nf-kb suggested that a gene fusion event created the nfkb genes in insects and verte‐ brates (Sullivan et al., 2007). This is consistent with the hypothesis that interactions between transcription factors of the Rel members and members of the IκB gene family evolved to reg‐ ulate genes mainly involved in immune inflammatory responses (Bonizzi & Karin, 2004).

NF-kappaB represents an ancient, generalized signaling system that has been co-opted for immune system roles independently in vertebrate and insect lineages (Friedman & Hughes, 2002). Therefore, while these proteins share a basic three-dimensional structure as predicted by their shared ankyrin repeat pattern and sequence, a possible evolutionary scenario based on this phylogenetic tree could be that subtle differences in the amino acid substitutions in the ankyrin repeats and flanking sequences occurred throughout evolution, which contrib‐ uted to their specificity of interaction with various members of the Rel family.
