**5. Detection of vulture TLR1 and IκBα expression in tissues**

In order to better understand the biological roles of TLR1 and IκBα, we analyzed their tissue expression pattern. The presence of transcripts encoding vulture TLR1 and IκBα in tissues was determined by real time RT-PCR. Biological samples were collected from vultures (about 8-10 months old) that were provisionally captive at the Centre for Wild Life Protec‐ tion, "El Chaparrillo", Ciudad Real, Spain. Blood was obtained by puncture of the branquial vein, located in the internal face of the wing, and collected in 10 ml tubes with EDTA as anticoagulant. Blood (10 ml) was diluted 1:1 (vol:vol) with PBS (Sigma) and the mononuclear fraction containing PBMC was obtained by density gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). All vulture tissues used for cDNA preparation were obtained fresh from euthanised birds that were impossible to recover.

RT-PCR was performed on a SmartCycler® II thermal cycler (Cepheid, Sunnyvale, CA, USA) using the QuantiTect® SYBR® Green RT-PCR Kit (Quiagen, Valencia, CA, USA), fol‐ lowing the recommendations of the manufacturer. We used primers GfTLR-Fw (5'-GCT TGC CAG TCA ACA TCA GA-3') and GfTLR-Rv (5'-GAA CTC CAG CGA CGT AAA GC-3'), which amplify a fragment of 158 bp of vulture TLR1 and primers IκBα -L (5'- CTG CAG GCA ACC AAC TAC AA -3') and IκBα –R (5'- TGA ATT CTG CAG GTC GAC AG-3'), which amplify a fragment of 165 b of vulture IκBα. Cycling conditions were: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, for 40 cycles. As an internal control, RT-PCR was performed on the same RNAs using the primers BA-Fw (5'-CTA TCC AGG CTG TGC TGT CC-3') and BA-Rv (5'-TGA GGT AGT CTG TCA GGT CAG G-3'), which amplify a fragment of 165 bp from the conserved housekeeping gene beta-actin. Control reactions were done using the same procedures, but without RT to control for DNA contamination in the RNA prepara‐ tions, and without RNA added to control contamination of the PCR reaction. Amplification efficiencies were validated and normalized against vulture beta actin, (GenBank accession number DQ507221) using the comparative Ct method. Experiments were repeated for at least three times with similar results. Tissues used for the study were artery, liver, lung, bur‐ sa cloacalis, heart, small intestine, peripheral blood mononuclear cells (PBMC), large intes‐ tine and kidney.

remained conserved because of their important structural and functional roles in regulating

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

Compared with other species, vulture IκBα exhibited the lowest number of predicted SU‐

**Structural feature** *G fulvus G gallus H sapiens S scrofa R norvegicus M musculus* **Amino acid residues** 313 318 317 314 314 314 *Number of ankyrin repeats* 5 5 5 5 5 5 *Phosphorylation sites* 14 13 14 15 14 13 *Predicted MW(KDa)* 35,17 35,40 35,61 35,23 35,02 35,02 *SUMOlation sites* 2 3 4 4 5 5

**Table 3.** Structural features of IκBα from Griffon vulture (*G. fulvus*), Chicken (*G. gallus*), human (*H. sapiens*), pig (*S. scrofa*), rat (*R. norvegicus*) and mouse (*M. musculus*) amino acid sequences. The theoretical molecular weight, number of ankyrin repeats, SUMOlation and of phosphorylation sites was calculated using the software available at the expasy web server (http://www.expasy.org). Genbank or Swiss accession number for proteins are EU161944 (*G. fulvus*), Q91974 (*G. gallus*), P25963 (*H. sapiens*), Q08353 (*S. scrofa*), Q63746 (*R. norvegicu*s), and Q9Z1E3 (*M. musculus*).

The comparison of the deduced amino acid sequence of vulture IκBα with the sequence of chicken, human, mouse, pig, and rat IκBα indicated that the deduced protein had a higher degree of similarity to chicken (91% of amino acid similarity) than to human (73%), mouse (74%), pig (73%) and rat (73%) sequences (Fig. 7). The analysis of the vulture IκBα sequence using the software NetPhos 2.0 (cita) revealed 14 potential phosphorylation sites: 10 Ser (S35, S39, S89, S160, S251, S263, S282, S287, S288, and S290), 1 Thr (T295) and 3 Tyr (Y16, Y45, and Y301). Although many of these residues were conserved in the aligned sequences from chicken, human, mouse, pig and rat IκBα, two phosphorylation sites (Y16 and S160) were

In order to better understand the biological roles of TLR1 and IκBα, we analyzed their tissue expression pattern. The presence of transcripts encoding vulture TLR1 and IκBα in tissues was determined by real time RT-PCR. Biological samples were collected from vultures (about 8-10 months old) that were provisionally captive at the Centre for Wild Life Protec‐ tion, "El Chaparrillo", Ciudad Real, Spain. Blood was obtained by puncture of the branquial vein, located in the internal face of the wing, and collected in 10 ml tubes with EDTA as anticoagulant. Blood (10 ml) was diluted 1:1 (vol:vol) with PBS (Sigma) and the mononuclear fraction containing PBMC was obtained by density gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway). All vulture tissues used for cDNA preparation were obtained

*4.2.1. Amino acid sequence comparison of vulture IκBα with other species*

**5. Detection of vulture TLR1 and IκBα expression in tissues**

fresh from euthanised birds that were impossible to recover.

distinctive to the vulture sequence (Fig.8).

NF-κB.

Applications

256

MOlation sites (Table 3).

The level of TLR1 mRNA was higher in kidney, small intestine and PBMC (Fig. 9).

**Figure 9.** Relative expression of TLR1 and IκBα mRNA transcripts in vulture cells and tissues.

Real time RT-PCR was used to examine the relative amount of TLR1 (right) and IκBα (left) transcripts in vulture cells and tissues. The data were normalised using the beta-actin gene and calculated by the delta Ct method.

Moderate vulture TLR1 mRNA levels were observed in Bursa cloacalis and large intestine, whereas the lowest TLR1 mRNA levels were found in liver, heart and artery (Fig. 9). It has been reported that the patterns of TLR tissue expression are variable, even among closely related species (Zarember & Godowski 2002). Likewise, the intensity and the anatomic loca‐ 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‐ ble to microorganisms upon infection.

used for the phylogenetic tree were Q91974, P25963, Q08353, Q63746, Q1ET75, Q6DCW3,

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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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

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

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).

Q8WNW7, Q6K196, Q1ET75, Q8QFQ0 and Q9Z1E3, respectively.

al., 2006).

species.

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 inhibitor of the TLR-signalling pathway.
